Insulin Modulates Intracellular Apolipoprotein B mRNA ... · ii . Insulin Modulates Intracellular Apolipoprotein B mRNA Traffic into RNA Granules/ Cytoplasmic P Bodies: Implications
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Insulin Modulates Intracellular Apolipoprotein B mRNA Traffic
into RNA Granules/Cytoplasmic P Bodies: Implications in
Translational Control
by
Navaz Karimian Pour
A thesis submitted in conformity with the requirements for the degree of Master of Science
Insulin Modulates Intracellular Apolipoprotein B mRNA Traffic into RNA Granules/ Cytoplasmic P Bodies: Implications in Translational Control
Navaz Karimian Pour
Master of Biochemistry
Department of Biochemistry University of Toronto
2012
Abstract
Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level;
however, the molecular mechanisms that govern translational control of apoB mRNA
remains largely unknown. We imaged intracellular apoB mRNA traffic and determined
whether insulin silences apoB mRNA translation by trafficking into translationally-silenced
cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by
using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA
sequence that binds MS2 protein. We observed a statistically significant increase in the
localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely,
acute insulin treatment (1h) did not show any significant effect. Insulin was also found to
reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells.
Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2
cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA
pools.
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Table of Contents
Abstract .................................................................................................................................................. ii
Table of Contents .................................................................................................................................. iii
List of Figures ....................................................................................................................................... vi
List of Tables....................................................................................................................................... viii
List of abbreviations .............................................................................................................................. ix
I. Introduction ......................................................................................................................................... 1
1.1 Introduction to Lipoproteins......................................................................................................... 1
1.2 Apolipoprotein B .......................................................................................................................... 1
1.2.1 The Importance of Studying ApoB ....................................................................................... 2
1.2.2 Structural Features of Human ApoB Gene, mRNA, and Protein .......................................... 3
1.2.3 ApoB Protein Function and Importance ................................................................................ 6
1.2.4 Hepatic Regulation of ApoB Gene Expression ..................................................................... 6
1.3 Role of Insulin in Metabolic Regulation of ApoB ..................................................................... 12
1.3.1 Insulin Signaling Pathways Involved in ApoB Biosynthesis and Regulation ..................... 13
1.3.2 ApoB Overproduction in Insulin Resistance States (Type 2 Diabetes and Obesity) .......... 14
1.4 The Process of mRNA Translation ............................................................................................ 15
2.2.17 Statistical Analysis of Imaging Data ................................................................................. 40
III. Results ............................................................................................................................................ 41
3.1.1 Results of Constructing a Linear Sucrose Gradient ............................................................ 41
3.1.2 Effect of Insulin on apoB mRNA Translation ..................................................................... 42
3.1.3 Under conditions that inhibit apoB mRNA polysomal association, insulin acutely stimulates global translation ......................................................................................................... 46
3.1.4 Effect of Insulin on Beta-2-microglobulin mRNA Distribution (as a control) ................... 47
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3.1.5 Determination of ApoB mRNA Abundance and Optimization of Real Time PCR Experiments using Internal Controls ............................................................................................ 49
3.1.6 Effect of Acute Insulin Treatments on ApoB mRNA Translation (Data Normalized to Internal Control Gene) ................................................................................................................. 52
3.1.7 Effect of Chronic Insulin Treatments on ApoB mRNA Translation (Data Normalized to Internal Control Genes) ................................................................................................................ 54
3.1.8 Effect of Insulin on Polysomal Distribution of Beta-2-microglobulin mRNA ................... 56
3.2 Translational Control of ApoB mRNA: Role of Cytoplasmic Ribonucleoproteins ................... 57
3.2.1 Construction of pCMV-MS2bs-24X-cyto Plasmid ............................................................. 57
3.2.2 Construction of pCMV-5’UTR-ApoB15%-3’UTR-MS2bs-24X-cyto Chimeric Plasmid .. 60
3.2.3 Detection of P Bodies (PBs) in HepG2 Cells and the Influence of Puromycin on PB formation ...................................................................................................................................... 62
3.2.4 Employing the MS2 Tagging System to Visualize ApoB mRNA in HepG2 Cells ............ 65
3.2.5 Transfection of HepG2 Cells with pMS2-GFP-SV40 NLS Plasmid .................................. 66
3.2.6 Insulin Induces Co-localization of ApoB mRNA with P Bodies ........................................ 69
3.2.7 Insulin does not affect the Co-localization of Beta-Globin mRNA with P Bodies ............. 71
IV. Discussion ...................................................................................................................................... 77
4.2 Storage of ApoB mRNA in Cytoplasmic RNA Granules .......................................................... 80
4.3 Postulated Mechanism of Insulin Modulation of ApoB mRNA Traffic into P bodies .............. 85
V. Conclusions ..................................................................................................................................... 88
VI. Future Directions ........................................................................................................................... 90
Reference List ...................................................................................................................................... 91
Figure 1 Schematic diagram of the pentapartite structural model of apoB and a three-dimensional consensus
model for LDL ............................................................................................................................................... 5
Figure 2 Synthesis of a linear sucrose gradient. ................................................................................................... 42
Figure 3 Effect of short-term insulin treatment on apoB mRNA association with polysomes............................. 44
Figure 4 Effect of long-term insulin treatment on apoB mRNA association with polysomes ............................ 45
Figure 5 Insulin acutely stimulates global mRNA translation in HepG2 cells. .................................................... 47
Figure 6 Effect of long-term insulin treatment on Beta-2-microglobulin mRNA (positive control) association
with polysomes ............................................................................................................................................ 48
Figure 7 Validation of potential internal control genes ........................................................................................ 51
Figure 8 Effect of short-term insulin treatment on apoB mRNA association with polysomes (data normalized to
internal control genes) .................................................................................................................................. 53
Figure 9 Effect of long-term insulin treatment on apoB mRNA association with polysomes (data normalized to
internal control genes) .................................................................................................................................. 55
Figure 10 Effect of long-term insulin treatment on B2M mRNA (positive control) association with polysomes
(Data normalized to internal control genes) ................................................................................................. 56
Figure 11 Construction of pCMV-MS2bs-24X-cyto plasmid. ............................................................................. 59
Figure 12 Construction of pCMV-5’UTR-ApoB15%-3’UTR-MS2bs-24X-cyto plasmid. .................................. 61
Figure 13 Detection of P bodies in the cytoplasm of HepG2 cells. ...................................................................... 63
Figure 14 Effect of puromycin on P body formation ........................................................................................... 64
Figure 15 Construction map of MS2-GFP and apoB mRNA reporter plasmids .................................................. 66
Figure 16 Expression of pMS2-GFP-SV40 NLS vector and the effect of NLS. .................................................. 68
Figure 17 Visualizing apoB mRNA traffic in HepG2 Cells: Long-term exposure to insulin induced co-
localization of apoB mRNA with P bodies .................................................................................................. 70
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Figure 18 Visualizing apoB mRNA traffic in HepG2 Cells: Short term exposure to insulin did not induce co-
localization of apoB mRNA with P bodies .................................................................................................. 71
Figure 19 Effect of insulin on the co-localization of beta-globin mRNA with P bodies ...................................... 74
Figure 20 Quantification of apoB mRNA co-localization with P bodies ............................................................. 76
Figure 21 Quantification of beta-globin mRNA co-localization with P bodies ................................................... 76
Figure 22. A proposed model for insulin modulation of apoB mRNA traffic into P bodies ................................ 87
Figure 23 Visualizing apoB mRNA traffic in HepG2 Cells: 4 hour exposure to insulin induced co-localization
of apoB mRNA with P bodies. ................................................................................................................... 104
Figure 24 Visualizing apoB mRNA traffic in HepG2 Cells: 8 hour exposure to insulin induced co-localization
of apoB mRNA with P bodies. ................................................................................................................... 105
Figure 25 Visualizing apoB mRNA traffic in HepG2 Cells: 16 hour exposure to insulin induced co-localization
of apoB mRNA with P bodies. ................................................................................................................... 106
Figure 26 Visualizing apoB mRNA traffic in HepG2 Cells: 1 hour exposure to insulin din not induce co-
localization of apoB mRNA with P bodies ................................................................................................ 107
Figure 27 Effect of serum starvation on the co-localization of apoB mRNA with P bodies. ............................. 108
Figure 28 Effect of serum starvation on the co-localization of beta-globin mRNA with P bodies .................... 109
Figure 29 Effect of 1hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. ......... 110
Figure 30 Effect of 4 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. ........ 111
Figure 31 Effect of 8 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. ........ 112
Figure 32 Effect of 16 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies ....... 113
viii
List of Tables
Table 1 Characteristics of Plasma Lipoproteins. .................................................................................................... 3
Table 2 Primers used in Real Time PCR reactions .............................................................................................. 30
Table 3 List of primers used in PCR reaction to generate proper restriction sites at both ends of the 5’UTR-
Table 4 Primers used for direct sequencing ......................................................................................................... 37
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List of abbreviations
Acronym Definition ACF apobec-1 complementation factor AMEM alpha modification of eagles medium APO Apolipiprotein ApoB apolipoprotein B APOBEC-1 ApoB mRNA editing catalytic subunit 1 ASH1 achaete-Scute Homologue-1 ATCC american Type Culture Collection B2M beta-2-microglobulin BiP binding immunoglobulin protein C/EBP CCAAT/enhancer binding protein Caco-2 human colonic adenocarcinoma cells CBP20 cap-binding protein 20 CCR4-NOT cDNA
carbon catabolite repression 4- Negative on TATA complementary DNA
CE cholesteryl ester CHX cyclohexamide CM chylomicrons COS CV-1 (simian) in Origin, and carrying the SV40 genetic material CPEB cytoplasmic polyadenylation element binding protein Cy3 Cyanine CYFIP1 cytoplasmic FMR1 interacting protein 1 DAPI 4',6-Diamidino-2-Phenylindole Dcp1a mRNA-decapping enzyme 1A DIC differential interference contrast DNA deoxyribonucleic acid DTT dithiothreitol Edc3 enhancer of mRNA-decapping protein 3 eEFs eukaryotic elongation factors eFG elongation factor G EGP contain eIF4E, eIF4G and Pab1 eIFs eukaryotic initiation factors ER endoplasmic reticulum ERAD endoplasmic reticulum associated protein degradation eRFs eukaryotic release factors FBS fetal bovine serum FMRP fragile X mental retardation 1 protein Foxa2 forkhead box protein A2 G3BP Ras-GAP SH3 domain binding protein GAPDH Glyceraldehyde 3-phosphate dehydrogenase
x
Acronym Definition GFP green fluorescent protein gp78 glycoprotein 78 GTP guanosine triphosphate hDcp1 human decapping protein 1 HDL high density lipoproteins HepG2 human hepatoma cell line HIV-1 human immunodeficiency virus type 1 HMBS hydroxymethyl-bilane synthase HNF hepatocyte nuclear factor HPRT1 Hypoxanthine phosphoribosyltransferase 1 hsps heat shock proteins IDL intermediate density lipoproteins IL-6 interleukin 6 IR insulin receptor IRES internal ribosomal entry site IRS insulin receptor substrate LB lysogeny broth LDL low density lipoprotein LDLR low density lipoprotein receptor Lsm1-7 like sm LUC Luciferase MAPK/ERK mitogen-activated protein kinase/extracellular signal regulated kinase mRNA messenger ribonucleic acid mRNP messenger ribonucleoprotein MTP microsomal triglyceride transfer protein Pab1 poly A binding protein PABP poly A binding protein P-body processing body PBs Processing Bodies PBS phosphate buffered saline PCR polymerase chain reaction PERPP post-ER pre-secretory proteolysis PI3-K phosphatidylinositol 3-kinase PKC protein kinase C PUFAs polyunsaturated fatty acids P-value probability value RNA Ribonucleic acid RRF ribosome recycling factor SGs Stress Granules SH3 Hepatocyte nuclear factor SMAD small ‘mothers against’ decapentaplegic SV40 Simian vacuolating virus 40 TG Triglyceride TGF-β Transforming growth factor beta
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Acronym Definition TNF tumor necrosis factor tRNA transfer RNA UBC ubiquitin C UTR untranslated regions UTRs untranslated regions VLDL very low density lipoprotein YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein
zeta polypeptide β2M Beta-2-microglobulin
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I. Introduction
1.1 Introduction to Lipoproteins
Lipoproteins are macromolecular complexes composed of proteins and lipids. They
transport insoluble lipids, such as cholesterol, steroid hormones, bile, and triglycerides
between tissues. These sphere-shaped particles contain a core of insoluble cholesteryl ester
and triglyceride encircled by apoproteins (protein component), amphipathic phospholipids,
and cholesterol (1). The interactions of lipoproteins with cell surface receptors are mediated
by the apolipoproteins. Lipoproteins are synthesized by the liver and intestine and based on
their sizes and densities are classified into five major groups: chylomicrons (CM), very low
density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density
lipoproteins (LDL), and high density lipoproteins (HDL) (Table 1) (2). The focus of this
thesis project was on apolipoprotein B (apoB) which is the major protein component of the
atherogenic LDL particle (2).
1.2 Apolipoprotein B
Apolipoprotein B is a large amphipathic protein that is synthesized by the liver and
the intestine. In humans full-length apolipoprotein B (apoB100) is expressed by the liver and
is the main scaffold protein constituent of cholesterol rich VLDL, and LDL. A truncated
form of apoB containing 48% of the full length (apoB48) is expressed by the small intestine
and is crucial for absorption of dietary fat and chylomicron formation (3). ApoB plays an
essential role in synthesis, secretion and trafficking of lipoproteins (4). Moreover, ApoB100
mediates the binding of LDL to LDL receptor (LDLR) and facilitates the plasma clearance of
LDL (5-7).
1.2.1 The Importance of Studying ApoB
Atherosclerosis is the major cause of cardiovascular disease world-wide, and there is
a positive correlation between the augmented levels of LDL and VLDL in the circulation and
the development of coronary heart disease. Accumulation of cholesterol-rich LDL particles
in the intima of arterial vessels is a critical underlying mechanism that occurs during early
stage of plaque formation in atherosclerosis (8). Since apoB is the essential structural protein
component of the atherogenic LDL and VLDL, overproduction of apoB containing
lipoproteins causes dyslipidemia followed by the development of atherosclerosis and
coronary artery heart disease (9). Therefore, studying the regulation of synthesis and
secretion of apoB has been of significant interest for many years.
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Table 1 Characteristics of Plasma Lipoproteins [Adapted from reference (2)]. TG: Triglyceride; CE: Cholesteryl Ester.
Lipoprotein
Calss
Density (g/mL) Size (nm)
Major lipids Major
Apoproteins
CM <0.93 100-500 Dietary TGs B48, C-II, E
VLDL 0.93-1.006 30-80 Endogenous
TGs
B100, CII, E
IDL 1.006-1.019 25-50 CEs and TGs B100, E
LDL 1.019-1.063 18-28 CEs B100
HDL 1.063-1.210 5-15 CEs A, C-II, E
1.2.2 Structural Features of Human ApoB Gene, mRNA, and
Protein
The human apoB gene is located on chromosome 2 and spans over 43 kb (10;11). It
consists of 29 exons and 28 introns (12), and is transcribed to a 14.121 kb mRNA. The
5’untraslated region (UTR) and the 3’UTR of the mRNA are composed of 128 nucleotides
and 304 nucleotides, respectively (13;14). The 5’UTR region has 76% GC content and is
predicted to be able to make stable secondary structure (15). ApoB is mostly expressed in the
liver and intestine although a low level expression has been detected in the heart (16-18).
Translation of apoB mRNA is very tissue specific: human liver generates a full length form
of apoB called apoB100 that contains 4536 amino acids including a 27 residue signal peptide
(19), whereas human enterocytes produce apoB48. ApoB48 consists of 48% of the full
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length apoB from the N-terminus (20). This truncated form of apoB is generated due to a
posttranscriptional editing process that converts a glutamine codon at amino acid 2153
(CAA) to a stop codon (UAA) as a result of a deamination reaction at nucleotide 6666
(21;22). The expected molecular mass of full length apoB is 514 kDa while its real molecular
weight is around 550 kD caused by posttranslational modifications such as glycosylation,
phosphorylation, and acylation (19;23-26).
ApoB is capable of recruiting lipids and functions as a lipid transporter vehicle in the
plasma; this is mainly due to its amphipathic polypeptide structure. Despite several attempts,
scientists have not been able to obtain a high resolution apoB structure. This is mostly
because of its hydrophobic makeup and large size (27-30). On the other hand, computational
studies, circular dichroism and infrared spectroscopy propose a pentapartite domain structure
(31). This model predicts a globular βα1 domain at the very N-terminus that extends the first
15-20% of the polypeptide. This domain plays a very important role in lipid acquisition
during the lipid assembly process and is followed by amphipathic β1 domain. This domain
spans over 20%-40% of the length of the protein. α2 domain is next which is an amphipathic
helix, continued by a β2 domain that has the LDLR binding affinity. α3 domain is located at
the very C-terminus end of the polypeptide (Figure1) (3;31;32).
5
A
B
Figure 1 Schematic diagram of the pentapartite structural model of apoB and a three-dimensional consensus model for LDL. A: Illustrates the pentapartite structural model of apoB100: NH2-βα1-β1-α2-β2-α3-COOH composed of alternating α helices and β sheets. B: Left figure demonstrates the anticipated distribution of lipids in an LDL particle. Surface phospholipids are shown in yellow, whereas, core lipid is in red. Green represents phospholipids, and amphipathic β sheets are in blue. Right figure represents the proposed organization of apoB100 on an LDL particle surface. β sheets and α helices are in blue and red, respectively [Adapted from reference (31)].
6
1.2.3 ApoB Protein Function and Importance
The most important function of apoB is to transport lipids and lipid soluble vitamins
from dietary fat and lipid storage tissues to tissues in demand. ApoB100 is also capable of
binding to LDL receptor (LDLR) and mediates endocytosis and plasma clearance of LDL.
Regulation of apoB synthesis is very crucial and both increased and reduced circulating
levels of apoB are known as risk factors for life threatening complications. For example, a
high plasma level of apoB is highly associated with dyslipidemia and increased risk of
cardiovascular disease. On the other hand, having too little apoB, observed in
hypobetalipoproteinemic patients, is linked to hepatic steatosis (33). In addition, apoB
knockdown is embryonic lethal (34;35).
1.2.4 Hepatic Regulation of ApoB Gene Expression
ApoB is constitutively expressed in the liver and shows relatively constant levels of
transcript under most metabolic stimuli (36-41). There is often more apoB protein
synthesized than secreted, signifying the importance of posttranslational degradative
mechanisms in regulating the secretion of this protein (42). Nonetheless, there is evidence
showing that apoB synthesis is regulated at the levels of transcription, translation, co-
translational proteasomal degradation and post-translational proteolysis.
7
1.2.4.1 Transcriptional Regulation of ApoB
Cell culture experiments using promoter reporter constructs revealed many cis-acting
sequences that play an important role in the regulation of apoB gene expression. For
example, the sequence spanning -150 and +124 of apoB promoter moderates gene expression
of the reporter in hepatic and intestinal cell lines (43-45). Some transcription factors that are
abundantly found in the liver such as, hepatic nuclear factor-3, -4 (HNF-3 and HNF-4) have
been found to bind to this region. In a study in HepG2 cells, Human hepatoma cell line, it
was shown that the region between +43 and +53 particularly up-regulated apoB gene
transcription (46;47) with a G nucleotide at +51 playing a crucial role (47). On the other
hand, the sequence extending between +20 and +40 appears to be important for down-
regulation of gene expression (46). In addition, it has been shown that some intronic regions,
such as a 443 bp segment within intron 2 and a 155 bp segment within intron 3 improve
apoB promoter activity in HepG2 and CaCo2, heterogeneous human epithelial colorectal
adenocarcinoma cells, cell lines (43-45).
Comparison of apoB gene expression data obtained from in vivo models to that
obtained from cell culture has shown some differences. For example, studies in transgenic
mice showed that the apoB promoter alone is inadequate to control apoB gene transcription
and that a 443 bp enhancer sequence within the second intron and also a fragment upstream
of the apoB promoter (-899 to -5262) are both essential for a high level of apoB expression
in the liver (48). Interestingly, the cis regulatory elements that modulate transcription of
apoB in the intestine are entirely different from their counterparts in the liver. For instant, the
second intron enhancer and the -899 to -5262 region are not important for human apoB gene
transcription in the liver (49). However, the 54-62 kb region upstream of the apoB gene plays
cytoplasmic P bodies (178). This plasmid was purchased from Dr. Nancy Kedersha’s
laboratory (Harvard Medical School, Brigham and Women’s Hospital, Boston, MA).
Finally, pEGFP-N1 plasmid (Clontech, Mountain View, CA). As opposed to pMS2-
GFP-NLS plasmid (third plasmid), this plasmid expresses GFP protein lacking nuclear
localization signal and was used as a control.
2.2.3 Transformation and Plasmid DNA Amplification
Subcloning efficiency DH5α competent cells (Invitrogen, Carlsbad, CA) were used to
amplify all plasmids. 50-100 μL of DH5α aliquots were thawed on wet ice. One μg of
plasmid DNA in water was then taken for transformation and added to the cells. PUC 19
plasmid (Invitrogen, Carlsbad, CA) was used as a positive control for transformation
reaction. Cells were mixed briefly with gentle tabbing on the tube and incubated on ice for
30 minutes. The heat shock reaction was performed by transferring cells to a 42°C water bath
for 30 seconds. Cells were then returned on ice. After 2 minutes, 900 μL of room
temperature LB Broth media without antibiotic was added, mixed, and incubated in a 37°C
incubator shaker (New Brunswick Scientific, Edison, NJ.), shaking at 250 rpm for 1 hour
(enough time for three growth cycles). After incubation, tubes were centrifuged for 5 minutes
at 5000 rpm at room temperature. All but 100 μL of the supernatant was discarded; pellet
was resuspended in the remaining of the supernatant and poured onto an LB-ampicillin agar
plate. A “hockey puck” spreader was used to spread the cells evenly over the plate. Plates
were placed in a 37°C incubator with the agar side up and the lid side down overnight.
Several colonies were picked off the plates using sterile pipettes and were individually
transferred to a 14 ml polypropylene falcon tubes (Becton Dickinson labware, NJ) containing
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3 mL LB Broth media and ampicilin (0.1 mg/ mL). Bacteria grew at 37°C for 8 hours in a
bacterial incubator shaker. After 8 hours, they were transferred to a new 250 mL LB broth
media containing the antibiotic, and grew overnight at 37°C in the bacterial shaker incubator.
To prepare a stock of bacteria, 850 µL of the growing bacteria was placed in a 1.5 mL
freezing tube (Sarstedt, Montreal, Quebec), and 150 µL of autoclaved glycerol (Sigma
Aldrich, St. Louis, MO) was added, vortexed, and kept on ice for 1 hour. Cells were then
stored at -80°C.
Midiprep plasmid isolation was conducted using the Endotoxin Free Plasmid
Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Plasmids were eluted using purified autoclaved water, and their concentrations were
determined by Nanodrop. The purity of the plasmid DNA was assessed by calculating the
ratio of optical density units at A260/A280, which was mostly around 1.8.
2.2.4 Restriction Enzyme Digestion
All restriction enzymes were purchased from New England Biolabs, Inc. (Ipswich,
MA). One unit of each enzyme was used to digest 1 μg of DNA for 3 hours at 37°C
according to the manufacturer’s protocols.
2.2.5 Gel extraction
Digested products were run on a 0.8% agarose gel (Invitrogen Life Technologies,
Grand Island, NY) at 50 V. Desired DNA bands were then cut and gel purified using
QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s
protocol.
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2.2.6 Ligation Reaction
Purified digested plasmids and insert fragments were mixed and incubated at 56°C
for 10 minutes to anneal. T4 DNA ligase reaction mixture (Invitrogen, Carlsbad, CA) was
then prepared according to the manufacturer’s protocol and added. Ligation reaction was
performed for 4 hours at 25οC and for some plasmids continued for another 48 hours at 16ο
C. T4 DNA Ligase enzyme was heat inactivated for 10 minutes at 65ο C. Half of the total
reaction (5 μL) was used to transform DH5α competent cells.
2.2.7 Construction of pCMV-MS2bs-24X-cyto Plasmid
The gene transcribing 24 tandem repeats of MS2bs from pSL-MS2bs-24X vector was
inserted into pCMV-myc-cyto plasmid using NcoI and NotI restriction enzymes and T4
DNA ligase (New England Biolabs).
2.2.8 Construction of pCMV-5’URT-apoB15%-3’UTR-MS2-24X-
cyto Chimeric Plasmid
To construct this plasmid, the 5’UTR-apoB15%-3’UTR sequence was obtained from
the modified pGL3 vector. In order to create proper restriction sites, 18 mer PCR primers
were designed (Table 3) to create an ApaI restriction site before the 5’UTR end and a BamHI
site after the 3’UTR end. A clamp of 3 random bases was added at both extreme ends of the
oligonucleotide primers to provide a sufficient grip for the restriction enzyme. PfuUltra II
fusion HS DNA polymerase enzyme (Stratagene, La Jolla, CA) was used to amplify the
sequence according to the manufacturer’s protocol (95°C, 2 minutes; 40 cycles (95°C 25
36
seconds; 62°C 25 seconds; 72°C 3 minutes); 72°C 15 minutes. This fragment was then
inserted in place on pCMV-MS2bs-24X-cyto plasmid.
Table 3 List of primers used in PCR reaction to generate proper restriction sites at both ends of the 5’UTR-apoB15%-3’UTR fragment. Primer Name Primer Sequence
5’ApaI-Forw AGAGGGCCCTTATTCCCACCGGGACCT
BamH13’-Rev AGAGGATCCCCGCCCCGACTCTAGATA
2.2.9 Direct DNA Sequencing
Direct DNA sequencing method was used to validate the sequence of constructed
plasmids (DNA sequencing facility, The Hospital for Sick Children, Toronto, ON)
(http://www.tcag.ca/dnaSequencingSynthesis.html). Table 4 represent a list of primers that
have been designed and used to sequence different constructs.
2.2.10 Transient Transfection Experiments
HepG2 cells were transiently co-transfected with 5.5 µg of pMS2-GFP-SV40 NLS
plasmid and 14.5 µg of either pCMV-5’URT-apoB15%-3’UTR-MS2-24X-cyto (reporter
plasmid) or EF1a-β-globin mRNA-MS2bs plasmid (control) using Lipofectamine 2000
reagent (Invitrogen, Carlsbad, CA) and reverse transfection method according to the
manufacturer’s recommendations; at about 50%- 60% confluency in a T75 flask HepG2 cells
were trypsinized, and mixed with the transfection complexes. Transfected HepG2 cells were
seeded on collagen-coated cover slips into six-well plates (8×105 cells/ well). Cells were
To make 100 collagen coated cover slips in a 6 well plate (100 μg/ well), 10 mg type
I collagen (MP Biomedicals, Solon, OH) was weighed and dissolved in 100 mL 0.05 M HCl
(0.1 mg/ mL collagen solution). The solution was then filtered using 0.45 μm filters (Sarstedt
Inc., Montreal, Quebec). One autoclaved cover slip (VWR, Mississauga, ON) was inserted
38
into each well and 1 mL of the solution was layerd on top. Collagen surface was air dried in a
cell culture hood overnight. Wells were washed twice with autoclaved H2O. Plates were air
dried in the hood, repacked, and stored at 4°C for up to 3 months.
2.2.12 GFP Expression and Insulin Treatment
Sixteen hours post transfection, GFP signal was detected under an Epifluorescence
Microscope (Zeis). At this time, cells were serum starved for half an hour and treated with
100 nM insulin (Eli Lilly, Canada Inc. Toronto, ON) for 1 h, 4 h, 8 h, or 16 h.
2.2.13 Immunostaining Experiments
To detect P bodies in HepG2 cells, human hepatoma cell line, that express exogenous
GFP, and to investigate the co-localization of P bodies with apoB mRNA, after proper
insulin treatments cells were fixed and stained for human enhancer of decapping large
subunit (hedls), a protein marker of P bodies. Cells were rinsed twice with PBS and fixed
immediately using 4% paraformaldehyde in 1XPBS solution for 15 minutes.
Paraformaldehyde was then aspirated and -20° C methanol was added for 10 minutes. Cells
were washed with 1XPBS, and blocked in 1X PBS containing 5% fetal bovine serum (FBS)
and 0.02% sodium azide (Sigma Aldrich, St. Louis, MO) for 1 hour. Mouse-anti-human GE-
1/hedls antibody (P70 S60 kinase α) (Santacruz Biotechnology, Inc., Santa Cruz, CA) was
diluted 1:1000 in the blocking solution and added to the fixed cells for 1 hour. After two
washes with 1XPBS (10 minutes each) Donkey-anti-mouse Rhodamine Red secondary
antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was diluted 1000
times in the blocking solution and added for one hour. Three washes with 1X PBS were
performed (10 minutes each), and cells were incubated with DAPI (4′6 -diamidino-2-
39
phenylindole·2HCl) (1 mg/ mL stock, 1:1000 dilution) (Santa Cruz Biotechnology, Inc.) for
15 minutes to stain the nucleus. After 3 washes with 1XPBS (10 minutes each) cells were
mounted in Dako flurescent mounting media (Dako North America, Inc., Carpinteria, CA),
slides were protected from light and kept at 4°C (179). All staining steps were performed on
a gentle shaker at room temperature.
2.2.14 Confocal Microscopy Imaging
Images were acquired from fixed cells using spinning disk confocal microscopy
(Zeiss) and velocity software. DAPI confocal, GFP confocal and Cy3 confocal laser channels
were used to detect the nucleus, GFP signal, and P bodies, respectively, with sensitivities
around 200 and exposures not longer that 1 second. Images were then deconvolved to
remove the out of focus light (90% similarity between the deconvolved image and the
original; 20 rounds of deconvolution).
2.2.15 Using hDCP1a, a Second Primary Antibody, to Detect P
bodies
To confirm the detection of P bodies another primary antibody, hDCP1a (human
decapping protein 1) (Santacruz Biotechnology, Inc., Santa Cruz, CA), was utilized in the
imaging experiments. This antibody targets, Dcp1a, another protein component of P bodies
(179).
2.2.16 Puromycin Treatment
Puromycin is known to disrupt the translational machinery and increase the size and
number of P bodies (180). To further validate the detection of P bodies, HepG2 cells were
40
treated with 1 mM puromycin dihydrochloride (Sigma Aldrich, St. Louis, MO) for 30
minutes to disrupt the translational machinery and promote P body formation. Cells were
then fixed and stained for P bodies.
2.2.17 Statistical Analysis of Imaging Data
Pearson correlation coefficient is a measure of the correlation (linear dependence)
between two variables X and Y, giving a value between 0 and 1. It is widely used as a
measure of the strength of linear dependence between two variables. We used this measure to
evaluate the strength of the co-localization of P bodies and apoB mRNA at different time
points. Velocity software version 5 was used to quantify the co-localization data and the
significance of the findings was validated by the Pearson’s correlation coefficient. Mean and
standard deviations (SD) of data were calculated and shown in graphs.
41
III. Results
3.1 Polysome Profiling
3.1.1 Results of Constructing a Linear Sucrose Gradient
In order to assess the insulin effect on the association of apoB mRNA with polysomes
cytoplasmic extracts of HepG2 cells were first subjected to sucrose gradient sedimentation.
To verify the linearity of the sucrose gradient Trypan Blue was premixed with the heavier
sucrose (55%) and the gradient was generated. The gradient was then fractionated and the
distribution of Trypan Blue was assessed by spectrophotometry. Absorbances of Trypan Blue
at 584 nm indicated a constant decrease in the concentration of Trypan Blur towards the top
of the gradient (Figure 2).
42
Figure 2 Synthesis of a linear sucrose gradient.A continuous sucrose gradient was generated using a 55% sucrose solution containing Trypan Blue and 12% sucrose solution lacking Trypan Blue. Eleven 1mL fractions were collected and spectrophotometry analysis was performed to verify the linearity of the sucrose gradient.
3.1.2 Effect of Insulin on apoB mRNA Translation
In order to investigate the effect of insulin on the translation of apoB mRNA,
polysomal and non polysomal pools were obtained using the sucrose fractionation method.
Real Time PCR was conducted to analyse the relative percentage of apoB mRNA in each
pool. HepG2 cells subjected to short-term and long-term insulin treatments were lysed and
fractionated on continuous sucrose gradients (12%-55%). Four 3 mL fractions were obtained
from each gradient. While the fraction on top contained monosomes and ribosomal subunits,
polysomes were found in the bottom layers. The forth fraction contained the heaviest
polysomes. Total RNA was then extracted from each fraction and subjected to Real Time
PCR analysis. The relative percentage of apoB mRNA in each fraction was calculated and
graphed. Data from four independent experiments suggested that very short term insulin
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treatments (15 minutes and 1 hour) increased apoB mRNA association with polysomes
(Figure 3), while long term insulin exposure (4 hours and 16 hours) showed an inhibitory
effect on the translation of apoB and shifted the mRNA towards the lighter polysomes and
monosome rich fractions (Figure 4).
44
Figure 3 Effect of short-term insulin treatment on apoB mRNA association with polysomes. HepG2 cells were treated with insulin for 15 minutes (on top) and 1 hour (on bottom). Cytoplasmic extracts were then subjected to density fractionation at 40000 rpm for 2.5 hours, and Real Time PCR was performed to measure the relative percentage of apoB mRNA in each fraction. (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient. Data shown is mean +/- SD. *= p< 0.05
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Figure 4 Effect of long-term insulin treatment on apoB mRNA association with polysomes. HepG2 cells were treated with insulin for 4 hours (on top) and 16 hours (on bottom). Cytoplasmic extracts were then subjected to density fractionation at 40000 rpm for 2.5 hours, and Real Time PCR was performed to measure the relative percentage of apoB mRNA in each fraction. (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient. Data shown is mean +/- SD. *= p< 0.05
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3.1.3 Under conditions that inhibit apoB mRNA polysomal
association, insulin acutely stimulates global translation
As a control, we also examined the insulin effect on global translation in HepG2 cells
by assaying the absorbance of fractions at 254 nm. As shown in Figure 5, short term insulin
treatment (15 minutes) promoted global translation, as depicted by the rightward shift of the
polysomal profile (indicating the formation of heavy polysomes), similar to its effect on
apoB mRNA. However, following 4 hours of insulin treatment, although apoB mRNA
association with polysomes was inhibited, global translation was noticeably increased as
indicated by the presence of heavy polysomes. After 16 hours, the effect of insulin on global
translation was lost, while its inhibition of apoB mRNA translation persisted (Figure 5).
47
Figure 5 Insulin acutely stimulates global mRNA translation in HepG2 cells. In order to assess the general effect of insulin on the cellular protein translation, HepG2 cells were serum starved briefly and insulin treated for 15 minutes, 1 h, 4 h, and 16 hours. Cytoplasmic extracts were then analyzed using polysome gradients. Gradients were then fractionated using the upward displacement method and the absorbance at 254 nm was continuously monitoried.
3.1.4 Effect of Insulin on Beta-2-microglobulin mRNA
Distribution (as a control)
We were able to show that long-term insulin treatments (4 hour and 16 hour)
decreased the association of apoB mRNA with polysomes and therefore has a negative effect
on the translation of this mRNA. In order to confirm that this is not a universal response to
insulin, we examined the effect of long term insulin exposure on beta-2-microglobulin
mRNA translation, and observed an increased association of this mRNA with the heaviest
polysomal fractions 16 hours post insulin treatment (Figure 6).
Figure 6 Effect of long-term insulin treatment on Beta-2-microglobulin mRNA (positive control) association with polysomes. HepG2 cells were insulin treated for 16 hours and the cytoplasmic extracts were analyzed using polysome gradients. Relative percentage of Beta-2-microglobulin mRNA in each fraction was then measured by Real Time PCR. (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient. Data shown is mean +/- SD. *= p< 0.05
Figure 7 Validation of potential internal control genes. HepG2 cells were insulin treated for 15 minutes (A), 1 hour (B), 4hours (C), and 16 hours (D). Cytoplasmic extracts were then analyzed using polysome gradients. Relative percentage of HMBS mRNA (A and B), B2M (C), and UBC (D) in each fraction was then measured by Real Time PCR. (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient. Data shown is mean +/- SD.
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52
3.1.6 Effect of Acute Insulin Treatments on ApoB mRNA
Translation (Data Normalized to Internal Control Gene)
The threshold cycle numbers obtained from Real Time PCR experiments in section
3.1.2 correspond to relative percentage of apoB mRNA in each fraction was then normalized
to the relative amounts of HMBS mRNAs. Results from four independent experiments were
analyzed and presented in Figure 8. Fifteen minutes after insulin treatment, similar to
unnormalized data, a significant amount of apoB mRNA bound to the heaviest polysome
fraction was observed which indicates an increase in apoB mRNA translation. Conversely, 1
hour insulin treatment shifted this mRNA towards the lighter fractions.
53
Figure 8 Effect of short-term insulin treatment on apoB mRNA association with polysomes (data normalized to internal control genes). HepG2 cells were treated with insulin for 15 minutes (on top) and 1 hour (on bottom). Cytoplasmic extracts were then analyzed using polysome gradients and Real Time PCR was performed to measure the relative percentage of apoB mRNA in each fraction. Relative percentages of apoB mRNA were normalized to HMBS mRNA amount (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient). Data shown is mean +/- SD. *= p< 0.05
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54
3.1.7 Effect of Chronic Insulin Treatments on ApoB mRNA
Translation (Data Normalized to Internal Control Genes)
As was explained in section 3.1.5 the amounts of B2M and UBC mRNAs remained
constant in the presence and absence of insulin 4h and 16h post treatment, respectively.
ApoB mRNA levels were normalized to these genes and the average results of four
independent experiments were analyzed (Figure 9). 4 hours post insulin treatment apoB
mRNA made a significant shift from translationally active mode to translationally inactive
form. Likewise more apoB mRNA was associated with lighter polysomes and monosome
rich fractions 16 hour post insulin treatment compared to the control.
55
Figure 9 Effect of long-term insulin treatment on apoB mRNA association with polysomes (data normalized to internal control genes). HepG2 cells were treated with insulin for 4 hours (on top) and 16 hour (on bottom). Cytoplasmic extracts were then analyzed using polysome gradients and Real Time PCR was performed to measure the relative percentage of apoB mRNA in each fraction. Relative percentages of apoB mRNA were normalized to B2M (top) and UBC (bottom) mRNA amounts (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient). Data shown is mean +/- SD. *= p< 0.05
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3.1.8 Effect of Insulin on Polysomal Distribution of Beta-2-
microglobulin mRNA
We were able to show that long-term insulin treatments shifted apoB mRNA towards
lighter polysomes or monosomal fractions. In order to confirm that this is not a global effect
of insulin on translation, the association of B2M mRNA with polysomes was evaluated in the
presence of insulin. Relative percentage of B2M mRNA level in each fraction was
normalized to UBC mRNA. UBC mRNA level remained constant in each fraction in the
presence and absence of insulin (Figure 10). B2M mRNA showed a significantly higher
association with the heaviest polysomal fraction in the presence of insulin compared to the
serum starved sample.
Figure 10 Effect of long-term insulin treatment on B2M mRNA (positive control) association with polysomes (Data normalized to internal control genes). HepG2 cells were treated with insulin for 16 hours. Cytoplasmic extracts were then analyzed using polysome gradients and Real Time PCR was performed to measure the relative percentage of apoB mRNA in each fraction. Relative percentages of apoB mRNA were normalized to UBC mRNA amount (Fraction 1 was the lightest top fraction and fraction 4 contained the heaviest polysomes at the bottom of the gradient). Data shown is mean +/- SD. *= p< 0.05
The role of P bodies was examined by applying a recently-established method that
has been successfully employed to visualize exogenously expressed mRNAs in mammalian
cells. We made use of a strong interaction between bacteriophage capsid MS2 protein and a
sequence specific RNA stem-loops structure. In this technique a fluorescent protein, for
example, GPF is fused to RNA phage MS2 coat protein with a nuclear localization signal at
the C terminus. The RNA of interest is constructed to contain tandem repeats of the specific
phage RNA sequence that binds MS2 coat protein. The strong interaction between the
bacteriophage capsid protein MS2 and the sequence specific RNA stem-loops structure helps
visualize the RNA
In order to visualize apoB mRNA with PBs, we constructed a chimeric plasmid
containing 15% of apoB gene with 5’ and 3’UTRs at both ends followed by 24 tandem
repeats of MS2 binding sites. HepG2 cells were then transiently co-transfected with this
chimeric plasmid and a plasmid expressing MS2 protein fused to GFP.
3.2.1 Construction of pCMV-MS2bs-24X-cyto Plasmid
In order to construct pCMV-MS2bs-24X-cyto plasmid, eukaryotic expression vector
pCMV-myc-cyto (Figure 11 A) and pSL-MS2bs-24X plasmid containing 24 tandem repeats
of MS2 binding sites (Figure 11 C) were double digested with NcoI and NotI enzymes. A
sequence containing 24 repeats of MS2 binding site (1482 bps) was then gel purified and
inserted into pCMV-myc-cyto plasmid (4855 bps) (Figure 11 B and 11 D). The sequence of
58
this newly synthesized plasmid named pCMV-MS2bs-24X-cyto was validated by direct
DNA sequencing.
59
C D
Figure 11 Construction of pCMV-MS2bs-24X-cyto plasmid. Both pCMV-myc-cyto (A) and pSL-MS2bs-24X vectors (C) were double digested with NcoI and NotI restriction enzymes (B and D). 24 repeats of MS2 binding site was then inserted into pCMV-myc-cyto vector with the help of T4 DNA ligase.
1Kb
mar
ker
A
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1482 bps
B
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1Kb
mar
ker
60
3.2.2 Construction of pCMV-5’UTR-ApoB15%-3’UTR-MS2bs-
24X-cyto Chimeric Plasmid
A graduate student in our laboratory had previously inserted a fragment of DNA
containing 5’untranslated region of apoB gene followed by a sequence encoding 15% of the
full length apoB from N-terminus and 3’untranslated region of apoB in a pGL3-Control
Vector (66) (Figure 12 A). In order to create pCMV-5’UTR-ApoB15%-3’UTR-MS2bs-24X-
cyto chimeric plasmid first PCR primers were designed to create new restriction sites at both
ends of the sequence (15% of the full length apoB enclosed by its UTRs). This fragment was
amplified (Figure 12 B) and ApaI and BamH1 restriction sites were added to the 5’ and 3’
end of the fragment, respectively. ApaI and BamH1 restriction enzymes were used to clone
this fragment into the previously synthesized pCMV-MS2bs-24X-cyto plasmid upstream of
the MS2 gene. The sequence of this plasmid was verified by direct DNA sequencing.
61
A
B
Figure 12 Construction of pCMV-5’UTR-ApoB15%-3’UTR-MS2bs-24X-cyto plasmid. A sequence containing 15% of the full length apoB enclosed by its UTRs was amplified (B) from a modified version of pGL3-Control Vector template (A) and positioned upstream of the MS2 gene using Apa1 and BamH1 restriction sites created by PCR (B).
5’UTR
3’UTR ApoB15%
2546 bps
62
3.2.3 Detection of P Bodies (PBs) in HepG2 Cells and the
Influence of Puromycin on PB formation
We used mouse-anti-human GE-1/hedls primary antibody to detect P bodies in
HepG2 cells. This antibody identifies a major protein component of P bodies called human
enhancer of decapping larger subunit. Donkey-anti-mouse Rhodamine Red conjugated
antibody was used to visualize the signal (Figure 13A). Immunostaining with GE-1/hedls
revealed ~60 foci per cell. A similar pattern was observed using an antibody against a second
P body protein, hDcp1a (human decapping protein 1) (Figure 13B). To further validate that
these are in fact P bodies, cells were subjected to 1 mM puromycin for 30 minutes (Figure
13C). Puromycin is known to promote the formation of P bodies by disrupting the
translational machinery. Results from 8 different slides indicated that puromycin
significantly increased P body count and size by 99% and 25%, respectively, (Figure 14) (P
< 0.005)
63
Figure 13 Detection of P bodies in the cytoplasm of HepG2 cells. HepG2 cells were fixed and immunostained for two protein components of P bodies, hedls (A), and hDcp1a (B), shown in red. DAPI was used to stain the nucleus, shown in blue. Puromycin was used to further verify the detection of P bodies (C), HepG2 cells were treated with 1 mM puromycin for half an hour, fixed and immunostained for hDCP1 (C). DIC (differential interference contrast) images of the same fields are represented on the right hand panel.
64
A
B
Figure 14 Effect of puromycin on P body formation. HepG2 cells were exposed to 1 mM puromycin for half an hour, then fixed and immunostained for hDCP1, one of the protein components of P bodies. The average size and number of P bodies were determined. Data shown is mean +/- SD. *= p < 0.005.
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65
3.2.4 Employing the MS2 Tagging System to Visualize ApoB
mRNA in HepG2 Cells
To use the MS2 tagging system to visualize apoB mRNA HepG2 cells were
transiently co-transfected with two plasmids (Figure 15): First, a chimeric DNA construct
that would transcribed to a reporter mRNA containing 15% of the full length apoB linked to
its UTRs (5’UTR-apoB15%-3’UTR) which was fused to MS2 binding site. The MS2 binding
site included 24 tandem repeats of 19 nucleotide RNA stem loop structures. This RNA is
recognizable by phage capsid MS2 protein which was encoded by the second plasmid. The
second plasmid made a MS2 protein fused to GFP with a nuclear localization signal at the C-
terminus end. Upon expression the GFP-MS2 coat protein binds the stem loop structure as a
dimer (183), This allows the detection of a specific RNA that is bound to the stem loop. The
presence of nuclear localization signal at the 3’ end of the MS2 protein helped the
elimination of false positive signals in the cytoplasm by sequestering any unbound MS2-GFP
in the nucleus.
66
Pol II promoter MS2 eGFP SV40 NLS
MS2-GFP Protein
PCMV promoter 5 ’UTR-apoB15-3’UTR PolyA signal
Repeats of MS2 binding sites
Reporter mRNA
Figure 15 Construction map of MS2-GFP and apoB mRNA reporter plasmids. A system composed of two plasmids enabled us to detect apoB mRNA in HepG2 cells. In this system one plasmid encoded a MS2 binding protein fused to GFP (top) and the other one was transcribed to a reporter mRNA containing part of apoB mRNA sequence followed by MS2 binding sites (bottom).
3.2.5 Transfection of HepG2 Cells with pMS2-GFP-SV40 NLS
Plasmid
In order to ensure that the green florescent in the cytoplasm only reflects the MS2-
GFP spicies that are associated with the reporter mRNAs via MS2 recognition sites and not
67
the free MS2 proteins, a nuclear localization signal (NLS) was positioned at the 3’ terminus
of this plasmid. HepG2 cells were transiently transfected with pMS2-GFP-SV40 NLS
plasmid. This plasmid encoded a green fluorescent protein that upon expression concentrated
in the nucleus due to the presence of a nuclear localization signal (Figure 16A).
As a control experiment, HepG2 cells were transfected with pEGFP-N1 plasmid that
expressed green florescent protein lacking the nuclear localization signal. In this case all GFP
signal was detected in the cytoplasm (Figure 16B).
68
A
B
Figure 16 Expression of pMS2-GFP-SV40 NLS vector and the effect of NLS. HepG2 cells were transfected with either pMS2-GFP-SV40 NLS (A) or pEGFP-N1 plasmids (B). 16 hours post transfection cells were fixed and immunostained for Dapi and imaged. In the presence of nuclear localization signal the GFP signal was sequestered in the nucleus (A), whereas, in the absence of a nuclear localization signal the GFP signal was dispersed in the cytoplasm (B).
69
3.2.6 Insulin Induces Co-localization of ApoB mRNA with P
Bodies
We transiently co-transfected HepG2 cells with pCMV-5’URT-ApoB15%-3’UTR-
MS2bs-24X-cyto and MS2-GFP-NLS constructs. Transfected cells were then treated with
insulin at different time points, fixed and immunostained for P body marker, GE-1/hedls.
Upon expression, MS2 binding proteins recognized their binding sites on the reporter
mRNAs as illustrated in Figure 16. Due to the presence of a nuclear localization signal
downstream of the MS2 protein any unbound MS2-GFP protein was sequestered in the
nucleus. We used spinning disk confocal microscopy to image the co-localization of apoB
mRNA with P bodies. Long-term insulin treatments (4 h, 8 h, and 16 h) (Figure 17)
significantly increased the co-localization of apoB mRNA with P bodies. On the other hand,
acute insulin treatment (1 h) (Figure 18) did not have any significant effect on the
localization of apoB mRNA with P bodies.
70
Figure 17 Visualizing apoB mRNA traffic in HepG2 Cells: Long-term exposure to insulin induced co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly and treated with insulin for 4 h (A), 8 h (B), and 16 h (C). Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue). Please see Appendix, figures 23-25 for full size confocal images.
71
Figure 18 Visualizing apoB mRNA traffic in HepG2 Cells: Short term exposure to insulin did not induce co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly (B) and treated with insulin for 1 h (A). Cells were then fixed and immunostained for hedls, one of the main protein components of P Bodies. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue). Please see Appendix, figures 26-27 for full size confocal images.
3.2.7 Insulin does not affect the Co-localization of Beta-Globin
mRNA with P Bodies
We were able to show that co-localization of apoB mRNA with P bodies increased at
4 hours, 8 hours and 16 hours post insulin treatment. To verify this is not the case for all
mRNAs we tested the influence of insulin on the co-localization of Beta-globin mRNA with
P bodies.
72
Beta-globin mRNA has already been shown to localize in P bodies (178). A plasmid
containing beta-globin mRNA fused to MS2 binding site and the MS2-GFP-NLS construct
were co-expressed in HepG2 cells. As shown in Figure 19, although there was marked co-
localization of beta-globin mRNA with the P body marker, no significant change was
observed following insulin treatment. Thus, in contrast to apoB mRNA, beta-globin mRNA
showed a high degree of co-localization with P bodies independent of insulin (Figure 19).
73
74
Figure 19 Effect of insulin on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly (A) and treated with insulin for 1 hour (B), 4 hours (C), 8 hours (D), and 16 hours (E). Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue). Please see Appendix, figures 28-32 for full size confocal images.
75
3.2.8 Statistical Analysis
Pearson correlation coefficient is a measure of the correlation (linear dependence)
between two variables and is widely used as a measure of the strength of linear dependence
between two variables. This measure was utilized in order to validate the strength of the co-
localization of P bodies and apoB mRNA under insulin stimuli.
In order to compare the co-localization of apoB mRNA with P bodies at different
time points of insulin treatment, Velocity software version 5 was used. Pearson’s correlation
coefficients were assessed based on the intensity of green and red pixels that overlapped as
an indication of the co-localization. A number between zero to one was assigned to each cell
with zero indicating no co-localization and one representing complete co-localization.
Numbers obtained from 8 different slides were averaged and graphed as represented in
Figure 20. These co-localization experiments allowed a qualitative assessment of the
association of intracellular apoB mRNA with P body granules. Our data suggestes that
insulin promotes apoB mRNA colocalization with P bodies after 4, 8, and 16 hours (Figure
20). Conversely, ß-globin mRNA shows a relatively high co-localization with P bodies at all
the experimental time points regardless of the presence and absence of insulin (Figure 21).
Figure 20 Quantification of apoB mRNA co-localization with P bodies. In order to evaluate the colocalization of apoB mRNA with P bodies Velocity software version 5 was used to measure the Pearson’s correlation coefficients. A number between zero and one was alloted to each cell with zero indicating no colocalization and one specifying perfect colocalization. Data shown is mean +/- SD. *= p< 0.05.
Figure 21 Quantification of beta-globin mRNA co-localization with P bodies.Velocity software version 5 was used to calculate Pearson’s correlation coefficients. A number between zero and one was given to each cell. Number one represents complete colocalization and zero shows no colocalization at all. Data shown is mean +/- SD.
0
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hour(s)
* * *
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77
IV. Discussion
Hepatic apoB synthesis is known to be regulated at multiple levels including
translation (40;41;63-65;164;182). Under different stimuli apoB mRNA levels stay relatively
constant while apoB protein synthesis and secretion varies greatly, thereby indicating the
importance of translational and posttranslational control mechanisms. Several studies from
our laboratory and others have shown that insulin reduces hepatic apoB protein synthesis and
secretion while mRNA levels remain stable (40;64;92;163;165). However, the molecular
mechanism(s) governing this inhibitory effect at the translational level have remained largely
unknown. Recent studies in our laboratory have revealed important translational mechanisms
of apoB mRNA involving the 5’ untranslated region (5’UTR). ApoB mRNA has a 5’UTR of
128 nucleotides and a 3’UTR of 304 nucleotides. Several structural features of apoB mRNA
UTR sequences suggest the presence of potential cis-elements that may interact with putative
trans-acting protein factors. A previous graduate student in our laboratory recently analyzed
the apoB UTR sequences using Mfold program, to predict RNA secondary structure, which
revealed elements within the 5' and 3'UTR’s of apoB mRNA with potential to form
secondary structure (66). They also assessed the biological activity of the putative RNA
motifs within the UTR sequences and found that 5'UTR motifs are important for optimal
translation of the apoB message. Deletion constructs of the UTR regions of apoB showed
that the 5'UTR was necessary and sufficient for insulin to inhibit apoB synthesis (66).
Interestingly, although insulin normally activates global translation of cellular protein
synthesis, it has a specific inhibitory effect on apoB mRNA translation. This suggests that
insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA
78
translation despite global translational stimulation. Our laboratory has now demonstrated that
insulin may modulate apoB mRNA translation via changes in the binding of a trans-acting
110-kDa protein factor to the 5’UTR (15). This putative RNA-binding protein (referred to as
p110) was found to specifically bind the 5’UTR of apoB mRNA, with its binding reduced in
the presence of insulin (15). Moreover, absence of insulin increased binding of this trans-
acting factor to the 5' UTR by 2-fold. Interestingly, translational control of apoB mRNA via
the 5’UTR and the binding of the 110 kDa protein factor was found to be regulated by
protein kinase C (PKC) signaling cascade (68). Using dual (bicistronic) luciferase constructs,
our laboratory also examined the role of internal ribosomal entry (IRES) with respect to the
5'UTR of the apoB mRNA and found that the apoB 5’UTR possesses IRES activity and basal
translational activity of the apoB mRNA may be partly cap independent (67). Presence of the
p110 protein highlights the importance of RNA-protein interactions that regulate the fate and
activity of apoB mRNA intracellularly.
There is now increasing evidence that eukaryotic mRNAs (particularly those with
longer half lives) exist in association with protein complexes in the form of RNA granules
which can govern both mRNA decay and translational activity. We thus investigated the
potential role of RNA granules in modulating apoB mRNA and its translational efficiency.
4.1 Polysome Profiling
In order to study the effect of insulin on apoB translation polysome gradient
sedimentation fractionation method was applied. This method is commonly used to study
translation under different physiological and experimental states. Our model system was
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HepG2 cells because they are hepatocellular carcinoma derived cell lines that constitutively
express apoB. We measured apoB mRNA levels associated with translationally-active
polysomes using Real-Time PCR. Our results suggested that insulin initially increases apoB
mRNA translation at 15 minutes and 1 hour, very similar to its effect observed on global
translation. Interestingly however, following longer insulin exposure (4 h), apoB mRNA
shifted towards lighter polysomes and monosome fractions, suggesting inhibition of
translational activity. At the same time point, global mRNA translation was still stimulated
by insulin treatment. With even longer insulin treatment (16 h), both apoB mRNA
association with heavy polysomes and total global polysomal activity were reduced. These
data suggest that apoB mRNA translation is uniquely inhibited by insulin under conditions
that stimulate global mRNA translation.
One of the challenges we were facing in this part of the project was to obtain enough
mRNA from each fraction to perform both the spectroscopic quantitation and Real Time
PCR. We first acquired twelve 1 mL fractions from each sucrose gradient, and used phenol
chloroform extraction method followed by lithium chloride (LiCl) precipitation to remove
the heparin which is a PCR inhibitor reagent. In order to eliminate the genomic DNA we
then performed DNase treatment. Finally in order to measure total mRNA in each fraction
we used 200 µL spectroscopy cuvettes. These steps resulted in significant loss of mRNA. In
order to address this problem, we fractionated the sucrose gradients to four 3 mL fractions,
and used a commercially available kit to extract higher amounts of RNA. We also made use
of a nanodrop spectrophotometer to measure the total RNA. In this type of
spectrophotometery one micro liter of the sample is sufficient to quantify RNA
concentration.
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4.2 Storage of ApoB mRNA in Cytoplasmic RNA
Granules
Mature mRNAs in the cytoplasm of eukaryotic cells are associated with a complex
network of ribonucleoprotein particles (mRNPs) (168;169). Early studies of mRNPs showed
the presence of two major proteins (170), a 70-kDa poly(A) binding protein (PABP) (171)
and a 50-kDa protein (p50) responsible for the repressed, nonactive state of mRNAs, such as
globin mRNA within free mRNP particles (172). Recent studies have identified a large
number of other protein components of mRNPs including RNA binding proteins, RNA
helicases, and translational factors (173). Importantly, RNA granules have been identified in
both germ cells and somatic cells that appear to play important roles in mRNA storage,
stability, and translational control. All RNA granules contain translationally silenced
mRNAs. New evidence suggests a dynamic interaction between these RNA granules and
translationally-active polysomal mRNAs (174), suggesting that the availability of some
mRNAs could be regulated by the rate of release from translationally-silenced mRNAs
within RNA granules. Stress granules (SG) contain mRNAs encoding most cellular proteins
and appear when translation initiation is impaired; for example, following exposure to
environmental stress (157). Another type of cytoplasmic RNA granule, called Processing
Bodies (PBs) are composed of RNA and proteins and translationally inactive mRNAs (149).
The protein components of P bodies are involved in RNA stability, storage, translation and
decay (183). P bodies are mainly composed of mRNAs, the 5’ to 3’ mRNA decay machinery,
the nonsense-mediated decay pathway proteins and the RNA- induced silencing complex
(183).
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It was initially unknown whether apoB mRNA translation can be controlled by the
release of translatable apoB mRNA transcripts from cytoplasmic stores of mRNPs that are
translationally repressed. However, the long half-life of apoB mRNA (16 h) suggests that a
significant proportion of the message may be stored in the cell prior to translation.
Interestingly, apoB mRNA polysome complexes have been reported to show unusual
physical properties and exhibit unique sedimentation behaviors more characteristic of
nonpolysomal mRNPs (175) further suggesting the existence of apoB mRNA in RNA
granules. We thus hypothesized that certain stimuli such as insulin may inhibit apoB mRNA
translation by reducing the release of translatable mRNA transcripts from stored mRNPs. In
order to test this hypothesis we investigated the colocalization of apoB mRNA with P bodies
under insulin stimuli.
The role of P bodies was examined by applying a recently-established method that
has been successfully employed to visualize exogenously expressed mRNAs in mammalian
cells (177). We made use of a strong interaction between bacteriophage capsid MS2 protein
and a sequence specific RNA stem-loops structure. Several studies have utilized this system
in order to track mRNA traffic in living cells. Bertrand et al., monitored the asymmetrical
movement of ASH1 mRNA in dividing yeast cells (184). Rook et al. and Fusco et al.
examined the movements of cytoplasmic RNA particles in neurons and COS cells (177;185).
Forrest and Gavis investigated the dynamic co-localization of endogenous nanos RNA in
Drosophila oocytes (186), and Kedersha et al. monitored the presence of single species of
mRNA transcripts in both SGs and PBs. This method has also been applied to study the RNA
trafficking in retroviruses (187;188). In this technique a fluorescent protein, for example,
GPF is fused to RNA phage MS2 coat protein with a nuclear localization signal at the C
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terminus (184). The RNA of interest is constructed to contain tandem repeats of the specific
phage RNA sequence that binds MS2 coat protein. The strong interaction between the
bacteriophage capsid protein MS2 and the sequence specific RNA stem-loops structure helps
visualize the RNA.
In order to visualize apoB mRNA with PBs, we constructed a chimeric plasmid
containing 15% of apoB gene with 5’ and 3’UTRs at both ends followed by 24 tandem
repeats of MS2 binding sites. HepG2 cells were then transiently co-transfected with this
chimeric plasmid and a plasmid expressing MS2 protein fused to GFP. The latter plasmid
contains a nuclear localization signal (NLS) at its C-terminus. Upon expression MS2 protein
finds its binding site on the reporter mRNA and any unbound MS2-GFP protein sequesters in
the nucleus due to the presence of the NLS.
Spinning disk confocal microscopy technique enabled us to visualize apoB mRNA in
fixed HepG2 cells. We then investigated the co-localization of exogenous apoB mRNA with
P bodies in the presence or absence of insulin at different time points. Confocal studies
revealed that long term insulin exposure promotes the co-localization of apoB mRNA with P
bodies with increases of 72% (after 4 h), 85% (after 8 h), and 89% (after 16 h) in PB co-
localization compared to respective non-insulin treated controls. However, a shorter (1 hour)
insulin treatment did not appear to induce observable changes in PB co-localization. As a
control, when HepG2 cells were transfected with pMS2-GFP-NLS plasmid alone, the entire
GFP signal was confined to the nucleus due to the presence of a nuclear localization signal at
the C-terminus of the protein.
It is important to note that although the above mentioned Singer’s method is a very
useful tool in imaging an exogenous mRNA in living cells it has some limitations. For
83
example to get a strong GFP signal presence of 24 repeats of MS2-binding sites is mandatory
(177) and it is not possible to get a good signal using fewer repeats of MS2 binding sites.
Since two molecules of GFP interact with each one of the stem-loop structures, a large
amount of GFP accumulates in a small area. This in fact increases the intensity of the signal
at the expense of lowering sensitivity.
Another alternative for us was to use in situ hybridization method in which the
presence of an mRNA could be assessed by designing probes that target specific sequences
on the mRNA of interest, apoB in our case. The in situ hybridization technique is more direct
and has a higher specificity compared to the method we used; however, it is not applicable to
live cells. Since we were planning to perform some live cell imaging analysis eventually and
look at the apoB mRNA movements under different experimental stimuli we chose to use the
former method.
Nancy Kedersha et al. had previously investigated the colocalization of β-globin
mRNA with P bodies and Stress Granules (178), and we thus used the same β-globin plasmid
as a control and examined the effect of insulin on the co-locolization of β-globin mRNA with
P bodies. HepG2 cells were transiently co-transfected with a plasmid containing β-globin
mRNA followed by the MS2-binding site and a plasmid expressing MS2 coat protein fused
to GFP followed by a nuclear localization signal. We observed clear co-localization of β-
globin mRNA with P bodies under normal cell culture condition. However, as opposed to
apoB mRNA, colocalization of β-globin mRNA with P bodies was insensitive to insulin in
HepG2 cells.
As can be observed from confocal images, apoB mRNA and PB marker did not co-
localize in all PBs. This is expected since each eukaryotic cell holds many P bodies
84
containing a variety of different kinds of mRNAs. A single PB does not contain all mRNAs,
and also a single species of mRNA does not exist in all P bodies. Also, in some of the images
we obtained, the GFP signal was observed in both the nucleus and the cytoplasm. This was
due to the large amount of MS2-GFP protein expression. Owing to the presence of the NLS,
the surplus GFP which was not bound to the mRNA sequestered in the nucleus. This in fact
was predictable from the onset and did not interfere with the interpretation of the imaging
data.
The monoclonal antibody used to recognize cytoplasmic P bodies showed some
nuclear staining in addition to the cytoplasm. This is due to the reactivity of this antibody
with p70 S6 kinase protein. The double specificity of this antibody was confirmed using
recombinant hedls, and this antibody was used to visualize PBs in p70 S6 kinases knockout
cells (179). The anti-hedls antibody readily detects cytoplasmic P bodies, and most of the
non-hedls signal is nuclear and did not impede the recognition of cytoplasmic P bodies (179).
We were able to show that puromycin, an aminonucleoside antibiotic derived from
the Streptomyces alboniger bacterium that causes premature chain termination during
translation, promoted the assembly of P bodies in HepG2 cells. Similar effect has been
reported by Nancy Kedersha et.al working on yeast, HeLa, and U2OS mammalian cell lines
(178).
In some of the images we acquired, GFP signal was detected in both the nucleus and
the cytoplasm. This was due to the presence of a large amount of MS2-GFP protein
expression. Owing to the existence of nuclear localization at the C termini of this protein the
surplus GFP which was not bound to the mRNA sequestered in the nucleus. This was in fact
predictable from the beginning and did not interfere with the accuracy of data interpretations.
and forcing the translationally-competent apoB mRNA towards P bodies for storage.
4.3 Postulated Mechanism of Insulin Modulation of ApoB
mRNA Traffic into P bodies
The present study shows that insulin silences apoB mRNA translation by localizing it
into P bodies and that this coincides with apoB mRNA run off from the translationally active
polysomes. However, the exact mechanism(s) that control apoB mRNA translational control
and the key molecule(s) that trigger apoB mRNA traffic into P bodies remain to be
elucidated. One possible mechanism may be through activation of an apoB mRNA binding
protein under insulin stimuli via protein modification(s), such as phosphorylation/
dephosphorylation. This activated RNA binding molecule will then interact with its proper
binding site on apoB mRNA, perhaps in the 5’UTR region that has been shown to play an
important role in regulating apoB mRNA translation in response to insulin. Such a cis-trans
interaction might result in the recruitment of P body factors to the mRNA through the
interaction of protein components of P bodies with the RNA binding protein(s) or directly
86
with apoB mRNA through new binding sites that appear after structural changes induced
following binding of the activated RNA binding factor. The newly formed small P Bodies
will then interact with each other through protein-protein interactions leading to the assembly
of larger P body aggregates. The translationally repressed apoB mRNA in P bodies could
have two faiths: it may either be degraded or reenter the translational machinery by
dissociating from P body granules, interacting with 40S ribosomal subunit and initiation
factors and assembeling into translationally active polysomes. Figure 22 depicts a proposed
model for insulin modulation of apoB mRNA localization in P bodies and the role of this
process in apoB mRNA translational control.
87
Figure 22. A proposed model for insulin modulation of apoB mRNA traffic into P bodies. Recent evidence suggest that insulin suppresses apoB mRNA translation through cis-transacting interactions with 5’UTR regions of apoB mRNA. Activation of RNA binding factor(s) by insulin facilitates their interaction with apoB mRNA and recruitment of P body components which nucleate the formation of larger P body aggregates. This results in translational suppression of apoB mRNA. Translationally repressed apoB mRNA could then either be degraded or disassembled from P bodies, re-associate with the translational machinery and be translated.
88
V. Conclusions
Although insulin normally activates global translation of cellular protein synthesis, it
has a specific inhibitory effect on apoB mRNA translation. This suggests that insulin induces
a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite
global translational stimulation. The mechanisms involved are post-transcriptional events
since apoB mRNA levels remained stable. In our investigation, we studied the potential role
of cytoplasmic RNA granules (P bodies) in insulin mediated translational regulation of apoB.
There is now increasing evidence that eukaryotic mRNAs (particularly those with longer half
lives) exist in association with protein complexes in the form of RNA granules which can
govern both mRNA decay and translational activity. P bodies control the translation of many
mRNAs in eukaryotic cells and we postulated that apoB mRNA is subcellularly
compartmentalized in the form of ribonucleoprotein complexes in RNA granules, which act
as a reservoir for translatable mRNA, a process potentially inhibitable by insulin.
In the current study we made use of a strong interaction between bacteriophage MS2
capsid protein and MS2 binding site to visualize apoB mRNA and to examine the influence
of insulin on the co-localization of apoB mRNA with P bodies. Spining disk confocal
imaging revealed that long-term insulin exposure promotes significant co-localization of
apoB mRNA with P bodies compared to non-insulin treated controls. Our data suggested that
insulin highly promotes apoB mRNA localization with P bodies after long-term insulin
treatment. Importantly, insulin-mediated co-localization of apoB mRNA with P bodies
coincided with alterations in translational activity of the message, based on polysomal
profiling experiments. Under these conditions, global mRNA translation was still stimulated
89
by insulin treatment. These data suggest that apoB mRNA translation is uniquely inhibited
by insulin under conditions that stimulate global mRNA translation. The time-course of
translational inhibition correlates with movement of apoB mRNA into cytoplasmic P bodies.
90
VI. Future Directions
ApoB mRNA translation is likely controlled by a complex network of RNA binding
proteins that interact with its 5’UTR and or 3’UTR regions. Insulin is a putative modulator of
some of these interactions. Therefore, it is important to investigate the effect of known RNA
binding proteins on apoB mRNA translation, and also to identify and characterize the novel
RNA binding proteins that regulate apoB mRNA translation. An important next step is to
study the regulatory effect of insulin on the cis-trans interactions on the apoB mRNA 5’UTR.
It will also be important to identify the RNA binding proteins involved in translational
repression of the apoB message and formation of RNA granules. Finally, considering the
exploration of interest in the role of miRNAs in regulating translation and mRNA decay, it
would be interesting to determine whether specific miRNAs are involved in translational
control of apoB mRNA. Recent bioinformatics analysis of the apoB mRNA sequence has
identified at least two miRNAs (miR-1202 and miR-544) that appear to interact with apoB
UTRs, supporting a possible role for miRNAs in regulation of apoB mRNA translation.
2. Tulenko,T.N. and Sumner,A.E. 2002. The physiology of lipoproteins. J.Nucl.Cardiol. 9:638-649.
3. Blasiole,D.A., Davis,R.A., and Attie,A.D. 2007. The physiological and molecular regulation of lipoprotein assembly and secretion. Mol.Biosyst. 3:608-619.
4. Brodsky,J.L. and Fisher,E.A. 2008. The many intersecting pathways underlying apolipoprotein B secretion and degradation. Trends Endocrinol.Metab 19:254-259.
5. Ejarque,I., Real,J.T., Martinez-Hervas,S., Chaves,F.J., Blesa,S., Garcia-Garcia,A.B., Millan,E., Ascaso,J.F., and Carmena,R. 2008. Evaluation of clinical diagnosis criteria of familial ligand defective apoB 100 and lipoprotein phenotype comparison between LDL receptor gene mutations affecting ligand-binding domain and the R3500Q mutation of the apoB gene in patients from a South European population. Transl.Res. 151:162-167.
6. Johnson,L.A., Altenburg,M.K., Walzem,R.L., Scanga,L.T., and Maeda,N. 2008. Absence of hyperlipidemia in LDL receptor-deficient mice having apolipoprotein B100 without the putative receptor-binding sequences. Arterioscler.Thromb.Vasc.Biol. 28:1745-1752.
7. Real,J.T., Chaves,F.J., Ejarque,I., Garcia-Garcia,A.B., Valldecabres,C., Ascaso,J.F., Armengod,M.E., and Carmena,R. 2003. Influence of LDL receptor gene mutations and the R3500Q mutation of the apoB gene on lipoprotein phenotype of familial hypercholesterolemic patients from a South European population. Eur.J.Hum.Genet. 11:959-965.
8. Hansson,G.K. and Libby,P. 2006. The immune response in atherosclerosis: a double-edged sword. Nat.Rev.Immunol. 6:508-519.
9. Avramoglu,R.K. and Adeli,K. 2004. Hepatic regulation of apolipoprotein B. Rev.Endocr.Metab Disord. 5:293-301.
10. Knott,T.J., Rall,S.C., Jr., Innerarity,T.L., Jacobson,S.F., Urdea,M.S., Levy-Wilson,B., Powell,L.M., Pease,R.J., Eddy,R., Nakai,H. et al. 1985. Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization. Science 230:37-43.
11. Law,S.W., Lackner,K.J., Hospattankar,A.V., Anchors,J.M., Sakaguchi,A.Y., Naylor,S.L., and Brewer,H.B., Jr. 1985. Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2. Proc.Natl.Acad.Sci.U.S.A 82:8340-8344.
12. Blackhart,B.D., Ludwig,E.M., Pierotti,V.R., Caiati,L., Onasch,M.A., Wallis,S.C., Powell,L., Pease,R., Knott,T.J., Chu,M.L. et al. 1986. Structure of the human apolipoprotein B gene. J.Biol.Chem. 261:15364-15367.
13. Knott,T.J., Wallis,S.C., Powell,L.M., Pease,R.J., Lusis,A.J., Blackhart,B., McCarthy,B.J., Mahley,R.W., Levy-Wilson,B., and Scott,J. 1986. Complete cDNA and derived protein sequence of human apolipoprotein B-100. Nucleic Acids Res. 14:7501-7503.
92
14. Taghibiglou,C., Carpentier,A., Van Iderstine,S.C., Chen,B., Rudy,D., Aiton,A., Lewis,G.F., and Adeli,K. 2000. Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model. J.Biol.Chem. 275:8416-8425.
15. Sidiropoulos,K.G., Pontrelli,L., and Adeli,K. 2005. Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein. Biochemistry 44:12572-12581.
16. Nielsen,L.B., Veniant,M., Boren,J., Raabe,M., Wong,J.S., Tam,C., Flynn,L., Vanni-Reyes,T., Gunn,M.D., Goldberg,I.J. et al. 1998. Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins. Circulation 98:13-16.
17. Nielsen,L.B., Bartels,E.D., and Bollano,E. 2002. Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice. J.Biol.Chem. 277:27014-27020.
18. Yokoyama,M., Yagyu,H., Hu,Y., Seo,T., Hirata,K., Homma,S., and Goldberg,I.J. 2004. Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse. J.Biol.Chem. 279:4204-4211.
19. Knott,T.J., Pease,R.J., Powell,L.M., Wallis,S.C., Rall,S.C., Jr., Innerarity,T.L., Blackhart,B., Taylor,W.H., Marcel,Y., Milne,R. et al. 1986. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 323:734-738.
20. Wang,A.B., Liu,D.P., and Liang,C.C. 2003. Regulation of human apolipoprotein B gene expression at multiple levels. Exp.Cell Res. 290:1-12.
21. Chen,S.H., Habib,G., Yang,C.Y., Gu,Z.W., Lee,B.R., Weng,S.A., Silberman,S.R., Cai,S.J., Deslypere,J.P., Rosseneu,M. et al. 1987. Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon. Science 238:363-366.
22. Powell,L.M., Wallis,S.C., Pease,R.J., Edwards,Y.H., Knott,T.J., and Scott,J. 1987. A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell 50:831-840.
23. Hoeg,J.M., Meng,M.S., Ronan,R., Demosky,S.J., Jr., Fairwell,T., and Brewer,H.B., Jr. 1988. Apolipoprotein B synthesized by Hep G2 cells undergoes fatty acid acylation. J.Lipid Res. 29:1215-1220.
24. Sparks,J.D., Sparks,C.E., Roncone,A.M., and Amatruda,J.M. 1988. Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL. J.Biol.Chem. 263:5001-5004.
25. Swift,L.L. 1996. Role of the Golgi apparatus in the phosphorylation of apolipoprotein B. J.Biol.Chem. 271:31491-31495.
26. Vukmirica,J., Nishimaki-Mogami,T., Tran,K., Shan,J., McLeod,R.S., Yuan,J., and Yao,Z. 2002. The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL. J.Lipid Res. 43:1496-1507.
93
27. Chatterton,J.E., Phillips,M.L., Curtiss,L.K., Milne,R., Fruchart,J.C., and Schumaker,V.N. 1995. Immunoelectron microscopy of low density lipoproteins yields a ribbon and bow model for the conformation of apolipoprotein B on the lipoprotein surface. J.Lipid Res. 36:2027-2037.
28. Gantz,D.L., Walsh,M.T., and Small,D.M. 2000. Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy. J.Lipid Res. 41:1464-1472.
29. Orlova,E.V., Sherman,M.B., Chiu,W., Mowri,H., Smith,L.C., and Gotto,A.M., Jr. 1999. Three-dimensional structure of low density lipoproteins by electron cryomicroscopy. Proc.Natl.Acad.Sci.U.S.A 96:8420-8425.
30. Spin,J.M. and Atkinson,D. 1995. Cryoelectron microscopy of low density lipoprotein in vitreous ice. Biophys.J. 68:2115-2123.
31. Segrest,J.P., Jones,M.K., De Loof,H., and Dashti,N. 2001. Structure of apolipoprotein B-100 in low density lipoproteins. J.Lipid Res. 42:1346-1367.
32. Fisher,E.A. and Ginsberg,H.N. 2002. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins. J.Biol.Chem. 277:17377-17380.
33. Tarugi,P., Averna,M., Di Leo,E., Cefalu,A.B., Noto,D., Magnolo,L., Cattin,L., Bertolini,S., and Calandra,S. 2007. Molecular diagnosis of hypobetalipoproteinemia: an ENID review. Atherosclerosis 195:e19-e27.
34. Farese,R.V., Jr., Ruland,S.L., Flynn,L.M., Stokowski,R.P., and Young,S.G. 1995. Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes. Proc.Natl.Acad.Sci.U.S.A 92:1774-1778.
35. Huang,L.S., Voyiaziakis,E., Markenson,D.F., Sokol,K.A., Hayek,T., and Breslow,J.L. 1995. apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J.Clin.Invest 96:2152-2161.
36. Dashti,N., Williams,D.L., and Alaupovic,P. 1989. Effects of oleate and insulin on the production rates and cellular mRNA concentrations of apolipoproteins in HepG2 cells. J.Lipid Res. 30:1365-1373.
37. Lin,M.C., Arbeeny,C., Bergquist,K., Kienzle,B., Gordon,D.A., and Wetterau,J.R. 1994. Cloning and regulation of hamster microsomal triglyceride transfer protein. The regulation is independent from that of other hepatic and intestinal proteins which participate in the transport of fatty acids and triglycerides. J.Biol.Chem. 269:29138-29145.
38. Moberly,J.B., Cole,T.G., Alpers,D.H., and Schonfeld,G. 1990. Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally. Biochim.Biophys.Acta 1042:70-80.
39. Nassir,F., Mazur,A., Serougne,C., Gueux,E., and Rayssiguier,Y. 1993. Hepatic apolipoprotein B synthesis in copper-deficient rats. FEBS Lett. 322:33-36.
40. Pullinger,C.R., North,J.D., Teng,B.B., Rifici,V.A., Ronhild de Brito,A.E., and Scott,J. 1989. The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life. J.Lipid Res. 30:1065-1077.
94
41. Sparks,J.D., Zolfaghari,R., Sparks,C.E., Smith,H.C., and Fisher,E.A. 1992. Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats. J.Clin.Invest 89:1418-1430.
42. Borchardt,R.A. and Davis,R.A. 1987. Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion. J.Biol.Chem. 262:16394-16402.
43. Kardassis,D., Laccotripe,M., Talianidis,I., and Zannis,V. 1996. Transcriptional regulation of the genes involved in lipoprotein transport. The role of proximal promoters and long-range regulatory elements and factors in apolipoprotein gene regulation. Hypertension 27:980-1008.
44. Zannis,V.I., Kan,H.Y., Kritis,A., Zanni,E., and Kardassis,D. 2001. Transcriptional regulation of the human apolipoprotein genes. Front Biosci. 6:D456-D504.
45. Zannis,V.I., Kan,H.Y., Kritis,A., Zanni,E.E., and Kardassis,D. 2001. Transcriptional regulatory mechanisms of the human apolipoprotein genes in vitro and in vivo. Curr.Opin.Lipidol. 12:181-207.
46. Chuang,S.S., Zhuang,H., Reisher,S.R., Feinstein,S.I., and Das,H.K. 1995. Transcriptional regulation of the apolipoprotein B-100 gene: identification of cis-acting elements in the first nontranslated exon of the human apolipoprotein B-100 gene. Biochem.Biophys.Res.Commun. 215:394-404.
47. Chuang,S.S. and Das,H.K. 1999. A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells. Biochim.Biophys.Acta 1436:600-605.
48. Brooks,A.R., Nagy,B.P., Taylor,S., Simonet,W.S., Taylor,J.M., and Levy-Wilson,B. 1994. Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice. Mol.Cell Biol. 14:2243-2256.
49. Antes,T.J., Goodart,S.A., Chen,W., and Levy-Wilson,B. 2001. Human apolipoprotein B gene intestinal control region. Biochemistry 40:6720-6730.
50. Nielsen,L.B., Kahn,D., Duell,T., Weier,H.U., Taylor,S., and Young,S.G. 1998. Apolipoprotein B gene expression in a series of human apolipoprotein B transgenic mice generated with recA-assisted restriction endonuclease cleavage-modified bacterial artificial chromosomes. An intestine-specific enhancer element is located between 54 and 62 kilobases 5' to the structural gene. J.Biol.Chem. 273:21800-21807.
51. Antes,T.J., Goodart,S.A., Huynh,C., Sullivan,M., Young,S.G., and Levy-Wilson,B. 2000. Identification and characterization of a 315-base pair enhancer, located more than 55 kilobases 5' of the apolipoprotein B gene, that confers expression in the intestine. J.Biol.Chem. 275:26637-26648.
52. Antes,T.J. and Levy-Wilson,B. 2001. HNF-3 beta, C/EBP beta, and HNF-4 act in synergy to enhance transcription of the human apolipoprotein B gene in intestinal cells. DNA Cell Biol. 20:67-74.
53. Singh,K., Batuman,O.A., Akman,H.O., Kedees,M.H., Vakil,V., and Hussain,M.M. 2002. Differential, tissue-specific, transcriptional regulation of apolipoprotein B secretion by transforming growth factor beta. J.Biol.Chem. 277:39515-39524.
54. Swagell,C.D., Henly,D.C., and Morris,C.P. 2007. Regulation of human hepatocyte gene expression by fatty acids. Biochem.Biophys.Res.Commun. 362:374-380.
95
55. Wang,Z. and Burke,P.A. 2007. Effects of hepatocyte nuclear factor-4alpha on the regulation of the hepatic acute phase response. J.Mol.Biol. 371:323-335.
56. Singh,K., Batuman,O.A., Akman,H.O., Kedees,M.H., Vakil,V., and Hussain,M.M. 2002. Differential, tissue-specific, transcriptional regulation of apolipoprotein B secretion by transforming growth factor beta. J.Biol.Chem. 277:39515-39524.
57. Greeve,J., Altkemper,I., Dieterich,J.H., Greten,H., and Windler,E. 1993. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins. J.Lipid Res. 34:1367-1383.
58. Hersberger,M., Patarroyo-White,S., Arnold,K.S., and Innerarity,T.L. 1999. Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element. J.Biol.Chem. 274:34590-34597.
59. Shah,R.R., Knott,T.J., Legros,J.E., Navaratnam,N., Greeve,J.C., and Scott,J. 1991. Sequence requirements for the editing of apolipoprotein B mRNA. J.Biol.Chem. 266:16301-16304.
60. Navaratnam,N., Fujino,T., Bayliss,J., Jarmuz,A., How,A., Richardson,N., Somasekaram,A., Bhattacharya,S., Carter,C., and Scott,J. 1998. Escherichia coli cytidine deaminase provides a molecular model for ApoB RNA editing and a mechanism for RNA substrate recognition. J.Mol.Biol. 275:695-714.
61. Mehta,A., Kinter,M.T., Sherman,N.E., and Driscoll,D.M. 2000. Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol.Cell Biol. 20:1846-1854.
62. Lellek,H., Kirsten,R., Diehl,I., Apostel,F., Buck,F., and Greeve,J. 2000. Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex. J.Biol.Chem. 275:19848-19856.
63. Lusis,A.J., Taylor,B.A., Quon,D., Zollman,S., and LeBoeuf,R.C. 1987. Genetic factors controlling structure and expression of apolipoproteins B and E in mice. J.Biol.Chem. 262:7594-7604.
64. Adeli,K. and Theriault,A. 1992. Insulin modulation of human apolipoprotein B mRNA translation: studies in an in vitro cell-free system from HepG2 cells. Biochem.Cell Biol. 70:1301-1312.
65. Sparks,J.D. and Sparks,C.E. 1990. Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes. J.Biol.Chem. 265:8854-8862.
66. Pontrelli,L., Sidiropoulos,K.G., and Adeli,K. 2004. Translational control of apolipoprotein B mRNA: regulation via cis elements in the 5' and 3' untranslated regions. Biochemistry 43:6734-6744.
67. Sidiropoulos,K.G., Meshkani,R., Avramoglu-Kohen,R., and Adeli,K. 2007. Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation. Arch.Biochem.Biophys. 465:380-388.
68. Sidiropoulos,K.G., Zastepa,A., and Adeli,K. 2007. Translational control of apolipoprotein B mRNA via insulin and the protein kinase C signaling cascades: evidence for modulation of RNA-protein interactions at the 5'UTR. Arch.Biochem.Biophys. 459:10-19.
96
69. Gordon,D.A., Jamil,H., Gregg,R.E., Olofsson,S.O., and Boren,J. 1996. Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J.Biol.Chem. 271:33047-33053.
70. Sniderman,A.D. and Cianflone,K. 1993. Substrate delivery as a determinant of hepatic apoB secretion. Arterioscler.Thromb. 13:629-636.
71. Yao,Z., Tran,K., and McLeod,R.S. 1997. Intracellular degradation of newly synthesized apolipoprotein B. J.Lipid Res. 38:1937-1953.
72. Bostrom,K., Wettesten,M., Boren,J., Bondjers,G., Wiklund,O., and Olofsson,S.O. 1986. Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. J.Biol.Chem. 261:13800-13806.
73. Borchardt,R.A. and Davis,R.A. 1987. Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion. J.Biol.Chem. 262:16394-16402.
74. Brodsky,J.L. 2007. The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation). Biochem.J. 404:353-363.
75. Nakatsukasa,K., Huyer,G., Michaelis,S., and Brodsky,J.L. 2008. Dissecting the ER-associated degradation of a misfolded polytopic membrane protein. Cell 132:101-112.
76. Liang,J.S., Kim,T., Fang,S., Yamaguchi,J., Weissman,A.M., Fisher,E.A., and Ginsberg,H.N. 2003. Overexpression of the tumor autocrine motility factor receptor Gp78, a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein B100 in HepG2 cells. J.Biol.Chem. 278:23984-23988.
77. Zhou,M., Fisher,E.A., and Ginsberg,H.N. 1998. Regulated Co-translational ubiquitination of apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory protein. J.Biol.Chem. 273:24649-24653.
78. Fisher,E.A., Zhou,M., Mitchell,D.M., Wu,X., Omura,S., Wang,H., Goldberg,A.L., and Ginsberg,H.N. 1997. The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70. J.Biol.Chem. 272:20427-20434.
79. Gusarova,V., Caplan,A.J., Brodsky,J.L., and Fisher,E.A. 2001. Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70. J.Biol.Chem. 276:24891-24900.
80. Qiu,W., Kohen-Avramoglu,R., Mhapsekar,S., Tsai,J., Austin,R.C., and Adeli,K. 2005. Glucosamine-induced endoplasmic reticulum stress promotes ApoB100 degradation: evidence for Grp78-mediated targeting to proteasomal degradation. Arterioscler.Thromb.Vasc.Biol. 25:571-577.
81. Wu,X., Sakata,N., Lele,K.M., Zhou,M., Jiang,H., and Ginsberg,H.N. 1997. A two-site model for ApoB degradation in HepG2 cells. J.Biol.Chem. 272:11575-11580.
82. Furukawa,S., Sakata,N., Ginsberg,H.N., and Dixon,J.L. 1992. Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells. J.Biol.Chem. 267:22630-22638.
83. Adeli,K. 1994. Regulated intracellular degradation of apolipoprotein B in semipermeable HepG2 cells. J.Biol.Chem. 269:9166-9175.
97
84. Fisher,E.A., Pan,M., Chen,X., Wu,X., Wang,H., Jamil,H., Sparks,J.D., and Williams,K.J. 2001. The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways. J.Biol.Chem. 276:27855-27863.
85. Pan,M., Cederbaum,A.I., Zhang,Y.L., Ginsberg,H.N., Williams,K.J., and Fisher,E.A. 2004. Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production. J.Clin.Invest 113:1277-1287.
86. Chirieac,D.V., Davidson,N.O., Sparks,C.E., and Sparks,J.D. 2006. PI3-kinase activity modulates apo B available for hepatic VLDL production in apobec-1-/- mice. Am.J.Physiol Gastrointest.Liver Physiol 291:G382-G388.
87. Galgani,J.E., Uauy,R.D., Aguirre,C.A., and Diaz,E.O. 2008. Effect of the dietary fat quality on insulin sensitivity. Br.J.Nutr. 100:471-479.
88. Riserus,U. 2008. Fatty acids and insulin sensitivity. Curr.Opin.Clin.Nutr.Metab Care 11:100-105.
89. Ding,W.X. and Yin,X.M. 2008. Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome. Autophagy. 4:141-150.
90. Fujita,E., Kouroku,Y., Isoai,A., Kumagai,H., Misutani,A., Matsuda,C., Hayashi,Y.K., and Momoi,T. 2007. Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum.Mol.Genet. 16:618-629.
91. Kruse,K.B., Brodsky,J.L., and McCracken,A.A. 2006. Autophagy: an ER protein quality control process. Autophagy. 2:135-137.
92. Adeli,K. and Sinkevitch,C. 1990. Secretion of apolipoprotein B in serum-free cultures of human hepatoma cell line, HepG2. FEBS Lett. 263:345-348.
93. Patsch,W., Franz,S., and Schonfeld,G. 1983. Role of insulin in lipoprotein secretion by cultured rat hepatocytes. J.Clin.Invest 71:1161-1174.
94. Patsch,W., Gotto,A.M., Jr., and Patsch,J.R. 1986. Effects of insulin on lipoprotein secretion in rat hepatocyte cultures. The role of the insulin receptor. J.Biol.Chem. 261:9603-9606.
95. Salhanick,A.I., Schwartz,S.I., and Amatruda,J.M. 1991. Insulin inhibits apolipoprotein B secretion in isolated human hepatocytes. Metabolism 40:275-279.
96. Sparks,C.E., Sparks,J.D., Bolognino,M., Salhanick,A., Strumph,P.S., and Amatruda,J.M. 1986. Insulin effects on apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat hepatocytes. Metabolism 35:1128-1136.
97. Brown,A.M. and Gibbons,G.F. 2001. Insulin inhibits the maturation phase of VLDL assembly via a phosphoinositide 3-kinase-mediated event. Arterioscler.Thromb.Vasc.Biol. 21:1656-1661.
98. Phung,T.L., Roncone,A., Jensen,K.L., Sparks,C.E., and Sparks,J.D. 1997. Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum. J.Biol.Chem. 272:30693-30702.
99. Sparks,J.D., Phung,T.L., Bolognino,M., and Sparks,C.E. 1996. Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase
98
activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes. Biochem.J. 313 ( Pt 2):567-574.
100. Taniguchi,C.M., Emanuelli,B., and Kahn,C.R. 2006. Critical nodes in signalling pathways: insights into insulin action. Nat.Rev.Mol.Cell Biol. 7:85-96.
101. Durrington,P.N., Newton,R.S., Weinstein,D.B., and Steinberg,D. 1982. Effects of insulin and glucose on very low density lipoprotein triglyceride secretion by cultured rat hepatocytes. J.Clin.Invest 70:63-73.
102. Borradaile,N.M., de Dreu,L.E., and Huff,M.W. 2003. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation. Diabetes 52:2554-2561.
103. Au,C.S., Wagner,A., Chong,T., Qiu,W., Sparks,J.D., and Adeli,K. 2004. Insulin regulates hepatic apolipoprotein B production independent of the mass or activity of Akt1/PKBalpha. Metabolism 53:228-235.
104. Allister,E.M., Borradaile,N.M., Edwards,J.Y., and Huff,M.W. 2005. Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes. Diabetes 54:1676-1683.
105. Au,W.S., Kung,H.F., and Lin,M.C. 2003. Regulation of microsomal triglyceride transfer protein gene by insulin in HepG2 cells: roles of MAPKerk and MAPKp38. Diabetes 52:1073-1080.
106. Wolfrum,C. and Stoffel,M. 2006. Coactivation of Foxa2 through Pgc-1beta promotes liver fatty acid oxidation and triglyceride/VLDL secretion. Cell Metab 3:99-110.
107. Taghibiglou,C., Carpentier,A., Van Iderstine,S.C., Chen,B., Rudy,D., Aiton,A., Lewis,G.F., and Adeli,K. 2000. Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model. J.Biol.Chem. 275:8416-8425.
108. Chirieac,D.V., Collins,H.L., Cianci,J., Sparks,J.D., and Sparks,C.E. 2004. Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats. Am.J.Physiol Endocrinol.Metab 287:E42-E49.
109. Bartels,E.D., Lauritsen,M., and Nielsen,L.B. 2002. Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice. Diabetes 51:1233-1239.
110. Biddinger,S.B. and Kahn,C.R. 2006. From mice to men: insights into the insulin resistance syndromes. Annu.Rev.Physiol 68:123-158.
111. Reaven,G.M. 1992. The role of insulin resistance and hyperinsulinemia in coronary heart disease. Metabolism 41:16-19.
112. Lewis,G.F. and Steiner,G. 1996. Acute effects of insulin in the control of VLDL production in humans. Implications for the insulin-resistant state. Diabetes Care 19:390-393.
114. Grundy,S.M., Mok,H.Y., Zech,L., Steinberg,D., and Berman,M. 1979. Transport of very low density lipoprotein triglycerides in varying degrees of obesity and hypertriglyceridemia. J.Clin.Invest 63:1274-1283.
115. Taskinen,M.R. 1995. Insulin resistance and lipoprotein metabolism. Curr.Opin.Lipidol. 6:153-160.
116. Reaven,G.M., Chen,Y.D., Jeppesen,J., Maheux,P., and Krauss,R.M. 1993. Insulin resistance and hyperinsulinemia in individuals with small, dense low density lipoprotein particles. J.Clin.Invest 92:141-146.
117. Lakka,H.M., Laaksonen,D.E., Lakka,T.A., Niskanen,L.K., Kumpusalo,E., Tuomilehto,J., and Salonen,J.T. 2002. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA 288:2709-2716.
118. Groppo,R. and Richter,J.D. 2009. Translational control from head to tail. Curr.Opin.Cell Biol. 21:444-451.
119. Majumdar,R., Bandyopadhyay,A., and Maitra,U. 2003. Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex. J.Biol.Chem. 278:6580-6587.
120. Lamphear,B.J., Kirchweger,R., Skern,T., and Rhoads,R.E. 1995. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J.Biol.Chem. 270:21975-21983.
121. Gingras,A.C., Raught,B., and Sonenberg,N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu.Rev.Biochem. 68:913-963.
122. Nakamura,Y. and Ito,K. 2003. Making sense of mimic in translation termination. Trends Biochem.Sci. 28:99-105.
123. Ramakrishnan,V. 2002. Ribosome structure and the mechanism of translation. Cell 108:557-572.
125. Petry,S., Weixlbaumer,A., and Ramakrishnan,V. 2008. The termination of translation. Curr.Opin.Struct.Biol. 18:70-77.
126. Sonenberg,N. and Hinnebusch,A.G. 2007. New modes of translational control in development, behavior, and disease. Mol.Cell 28:721-729.
127. Richter,J.D. and Sonenberg,N. 2005. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433:477-480.
128. Pyronnet,S., Dostie,J., and Sonenberg,N. 2001. Suppression of cap-dependent translation in mitosis. Genes Dev. 15:2083-2093.
129. Stebbins-Boaz,B., Cao,Q., de Moor,C.H., Mendez,R., and Richter,J.D. 1999. Maskin is a CPEB-associated factor that transiently interacts with elF-4E. Mol.Cell 4:1017-1027.
100
130. Jung,M.Y., Lorenz,L., and Richter,J.D. 2006. Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein. Mol.Cell Biol. 26:4277-4287.
131. Nakamura,A., Sato,K., and Hanyu-Nakamura,K. 2004. Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev.Cell 6:69-78.
132. Napoli,I., Mercaldo,V., Boyl,P.P., Eleuteri,B., Zalfa,F., De Rubeis,S., Di Marino,D., Mohr,E., Massimi,M., Falconi,M. et al. 2008. The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP. Cell 134:1042-1054.
133. Cho,P.F., Poulin,F., Cho-Park,Y.A., Cho-Park,I.B., Chicoine,J.D., Lasko,P., and Sonenberg,N. 2005. A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP. Cell 121:411-423.
134. Rom,E., Kim,H.C., Gingras,A.C., Marcotrigiano,J., Favre,D., Olsen,H., Burley,S.K., and Sonenberg,N. 1998. Cloning and characterization of 4EHP, a novel mammalian eIF4E-related cap-binding protein. J.Biol.Chem. 273:13104-13109.
135. Ostareck,D.H., Ostareck-Lederer,A., Shatsky,I.N., and Hentze,M.W. 2001. Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining. Cell 104:281-290.
136. Huttelmaier,S., Zenklusen,D., Lederer,M., Dictenberg,J., Lorenz,M., Meng,X., Bassell,G.J., Condeelis,J., and Singer,R.H. 2005. Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438:512-515.
137. Ceci,M., Gaviraghi,C., Gorrini,C., Sala,L.A., Offenhauser,N., Marchisio,P.C., and Biffo,S. 2003. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426:579-584.
138. Gandin,V., Miluzio,A., Barbieri,A.M., Beugnet,A., Kiyokawa,H., Marchisio,P.C., and Biffo,S. 2008. Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation. Nature 455:684-688.
139. Richter,J.D. and Klann,E. 2009. Making synaptic plasticity and memory last: mechanisms of translational regulation. Genes Dev. 23:1-11.
140. Sutton,M.A., Taylor,A.M., Ito,H.T., Pham,A., and Schuman,E.M. 2007. Postsynaptic decoding of neural activity: eEF2 as a biochemical sensor coupling miniature synaptic transmission to local protein synthesis. Neuron 55:648-661.
141. Sivan,G., Kedersha,N., and Elroy-Stein,O. 2007. Ribosomal slowdown mediates translational arrest during cellular division. Mol.Cell Biol. 27:6639-6646.
142. Collingridge,G.L., Isaac,J.T., and Wang,Y.T. 2004. Receptor trafficking and synaptic plasticity. Nat.Rev.Neurosci. 5:952-962.
143. Keiler,K.C., Waller,P.R., and Sauer,R.T. 1996. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271:990-993.
144. Haebel,P.W., Gutmann,S., and Ban,N. 2004. Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs. Curr.Opin.Struct.Biol. 14:58-65.
101
145. Akimitsu,N., Tanaka,J., and Pelletier,J. 2007. Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells. EMBO J. 26:2327-2338.
146. Rodriguez,A.J., Czaplinski,K., Condeelis,J.S., and Singer,R.H. 2008. Mechanisms and cellular roles of local protein synthesis in mammalian cells. Curr.Opin.Cell Biol. 20:144-149.
147. Sonenberg,N. 2008. eIF4E, the mRNA cap-binding protein: from basic discovery to translational research. Biochem.Cell Biol. 86:178-183.
148. Shyu,A.B., Wilkinson,M.F., and van Hoof,A. 2008. Messenger RNA regulation: to translate or to degrade. EMBO J. 27:471-481.
149. Anderson,P. and Kedersha,N. 2006. RNA granules. J.Cell Biol. 172:803-808.
150. Martin,K.C. and Ephrussi,A. 2009. mRNA localization: gene expression in the spatial dimension. Cell 136:719-730.
151. Anderson,P. and Kedersha,N. 2009. RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat.Rev.Mol.Cell Biol. 10:430-436.
152. Brengues,M. and Parker,R. 2007. Accumulation of polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-bodies in Saccharomyces cerevisiae. Mol.Biol.Cell 18:2592-2602.
153. Buchan,J.R., Muhlrad,D., and Parker,R. 2008. P bodies promote stress granule assembly in Saccharomyces cerevisiae. J.Cell Biol. 183:441-455.
154. Eulalio,A., Behm-Ansmant,I., and Izaurralde,E. 2007. P bodies: at the crossroads of post-transcriptional pathways. Nat.Rev.Mol.Cell Biol. 8:9-22.
155. Franks,T.M. and Lykke-Andersen,J. 2008. The control of mRNA decapping and P-body formation. Mol.Cell 32:605-615.
156. Gilks,N., Kedersha,N., Ayodele,M., Shen,L., Stoecklin,G., Dember,L.M., and Anderson,P. 2004. Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol.Biol.Cell 15:5383-5398.
157. Anderson,P. and Kedersha,N. 2008. Stress granules: the Tao of RNA triage. Trends Biochem.Sci. 33:141-150.
158. Tourriere,H., Chebli,K., Zekri,L., Courselaud,B., Blanchard,J.M., Bertrand,E., and Tazi,J. 2003. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J.Cell Biol. 160:823-831.
159. Kulkarni,M., Ozgur,S., and Stoecklin,G. 2010. On track with P-bodies. Biochem.Soc.Trans. 38:242-251.
160. Teixeira,D., Sheth,U., Valencia-Sanchez,M.A., Brengues,M., and Parker,R. 2005. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA. 11:371-382.
161. Liu,J., Valencia-Sanchez,M.A., Hannon,G.J., and Parker,R. 2005. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat.Cell Biol. 7:719-723.
162. Pillai,R.S., Bhattacharyya,S.N., Artus,C.G., Zoller,T., Cougot,N., Basyuk,E., Bertrand,E., and Filipowicz,W. 2005. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309:1573-1576.
102
163. Dashti,N., Williams,D.L., and Alaupovic,P. 1989. Effects of oleate and insulin on the production rates and cellular mRNA concentrations of apolipoproteins in HepG2 cells. J.Lipid Res. 30:1365-1373.
164. Theriault,A., Ogbonna,G., and Adeli,K. 1992. Thyroid hormone modulates apolipoprotein B gene expression in HepG2 cells. Biochem.Biophys.Res.Commun. 186:617-623.
165. Mohammadi,A., Theriault,A., and Adeli,K. 1996. In vitro translation and translocation of apolipoprotein B in a cell-free system from HepG2 cells. Biochem.Biophys.Res.Commun. 228:852-858.
166. Levy,E., Sinnett,D., Thibault,L., Nguyen,T.D., Delvin,E., and Menard,D. 1996. Insulin modulation of newly synthesized apolipoproteins B-100 and B-48 in human fetal intestine: gene expression and mRNA editing are not involved. FEBS Lett. 393:253-258.
167. Pan,M., Liang,J., Fisher,E.A., and Ginsberg,H.N. 2000. Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B. J.Biol.Chem. 275:27399-27405.
168. Keene,J.D. 2001. Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome. Proc.Natl.Acad.Sci.U.S.A 98:7018-7024.
169. Spirin,A.S. 1966. "Masked" forms of mRNA. Curr.Top.Dev.Biol. 1:1-38.
170. Jain,S.K., Pluskal,M.G., and Sarkar,S. 1979. Thermal chromatography of eukaryotic messenger ribonucleoprotein particles on oligo (dT)-cellulose. Evidence for common mRNA-associated proteins in various cell types. FEBS Lett. 97:84-90.
171. Blobel,G. 1973. A protein of molecular weight 78,000 bound to the polyadenylate region of eukaryotic messenger RNAs. Proc.Natl.Acad.Sci.U.S.A 70:924-928.
172. Minich,W.B., Maidebura,I.P., and Ovchinnikov,L.P. 1993. Purification and characterization of the major 50-kDa repressor protein from cytoplasmic mRNP of rabbit reticulocytes. Eur.J.Biochem. 212:633-638.
173. Angenstein,F., Evans,A.M., Ling,S.C., Settlage,R.E., Ficarro,S., Carrero-Martinez,F.A., Shabanowitz,J., Hunt,D.F., and Greenough,W.T. 2005. Proteomic characterization of messenger ribonucleoprotein complexes bound to nontranslated or translated poly(A) mRNAs in the rat cerebral cortex. J.Biol.Chem. 280:6496-6503.
174. Balagopal,V. and Parker,R. 2009. Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr.Opin.Cell Biol. 21:403-408.
175. Chen,X., Sparks,J.D., Yao,Z., and Fisher,E.A. 1993. Hepatic polysomes that contain apoprotein B mRNA have unusual physical properties. J.Biol.Chem. 268:21007-21013.
176. Bachand,F., Lackner,D.H., Bahler,J., and Silver,P.A. 2006. Autoregulation of ribosome biosynthesis by a translational response in fission yeast. Mol.Cell Biol. 26:1731-1742.
177. Fusco,D., Accornero,N., Lavoie,B., Shenoy,S.M., Blanchard,J.M., Singer,R.H., and Bertrand,E. 2003. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr.Biol. 13:161-167.
103
178. Kedersha,N., Stoecklin,G., Ayodele,M., Yacono,P., Lykke-Andersen,J., Fritzler,M.J., Scheuner,D., Kaufman,R.J., Golan,D.E., and Anderson,P. 2005. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J.Cell Biol. 169:871-884.
179. Kedersha,N. and Anderson,P. 2007. Mammalian stress granules and processing bodies. Methods Enzymol. 431:61-81.
180. Eulalio,A., Behm-Ansmant,I., Schweizer,D., and Izaurralde,E. 2007. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol.Cell Biol. 27:3970-3981.
181. Beckett,D. and Uhlenbeck,O.C. 1988. Ribonucleoprotein complexes of R17 coat protein and a translational operator analog. J.Mol.Biol. 204:927-938.
182. Yao,Z. and McLeod,R.S. 1994. Synthesis and secretion of hepatic apolipoprotein B-containing lipoproteins. Biochim.Biophys.Acta 1212:152-166.
183. Anderson,P. and Kedersha,N. 2006. RNA granules. J.Cell Biol. 172:803-808.
184. Bertrand,E., Chartrand,P., Schaefer,M., Shenoy,S.M., Singer,R.H., and Long,R.M. 1998. Localization of ASH1 mRNA particles in living yeast. Mol.Cell 2:437-445.
185. Rook,M.S., Lu,M., and Kosik,K.S. 2000. CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage. J.Neurosci. 20:6385-6393.
186. Forrest,K.M. and Gavis,E.R. 2003. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr.Biol. 13:1159-1168.
187. Basyuk,E., Boulon,S., Skou,P.F., Bertrand,E., and Vestergaard,R.S. 2005. The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs. J.Mol.Biol. 354:330-339.
188. Moore,M.D., Nikolaitchik,O.A., Chen,J., Hammarskjold,M.L., Rekosh,D., and Hu,W.S. 2009. Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses. PLoS.Pathog. 5:e1000627.
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Appendix
ApoB, Nucleus, P bodies
Figure 23 Visualizing apoB mRNA traffic in HepG2 Cells: 4 hour exposure to insulin induced co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly and treated with insulin for 4 h. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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ApoB, Nucleus, P bodies
Figure 24 Visualizing apoB mRNA traffic in HepG2 Cells: 8 hour exposure to insulin induced co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly and treated with insulin for 8 h. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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ApoB, Nucleus, P bodies
Figure 25 Visualizing apoB mRNA traffic in HepG2 Cells: 16 hour exposure to insulin induced co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly and treated with insulin for 16 h. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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ApoB, Nucleus, P bodies
Figure 26 Visualizing apoB mRNA traffic in HepG2 Cells: 1 hour exposure to insulin din not induce co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly and treated with insulin for 1 h. Cells were then fixed and immunostained for hedls, one of the main protein components of P Bodies. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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ApoB, Nucleus, P bodies
Figure 27 Effect of serum starvation on the co-localization of apoB mRNA with P bodies. HepG2 cells were transiently contransfected with pCMV-5’URT-ApoB15%-3’UTR-MS2bs-24X-cyto and MS2-GFP-NLS plasmids. 16 hours post transfection cells were serum sratved briefly. Cells were then fixed and immunostained for hedls, one of the main protein components of P Bodies. Green represents apoB mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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Beta-globin, Nucleus, P bodies
Figure 28 Effect of serum starvation on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly. Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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Beta-globin, Nucleus, P bodies
Figure 29 Effect of 1hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly and treated with insulin for 1 hour. Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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Beta-globin, Nucleus, P bodies
Figure 30 Effect of 4 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly and treated with insulin for 4 hours. Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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Beta-globin, Nucleus, P bodies
Figure 31 Effect of 8 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly and treated with insulin for 8 hours. Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).
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Beta-globin, Nucleus, P bodies
Figure 32 Effect of 16 hour insulin exposure on the co-localization of beta-globin mRNA with P bodies. HepG2 cells were co-transfected with EF1a-β-globin mRNA-MS2-bs and MS2-GFP-NLS plasmids, cells were then serum starved briefly and treated with insulin for 16 hours. Cells were then fixed and immunostained for hedls, one of the protein components of P bodies. Green represents beta-globin mRNA, P bodies are in red and DAPI was used to stain the nucleus (blue).