Investigation of the Ribosome Independent mRNA Localization to … · 2016-01-08 · Abstract Localization of mRNA to various subcellular compartments is a widespread phenomenon in
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i
Investigation of the Ribosome Independent mRNA Localization to
the Endoplasmic Reticulum
by
Xianying Amy Cui
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Biochemistry University of Toronto
3.3 mRNA Remains Associated to the ER Independently of Translation and Ribosome-
Association ...................................................................................................................................................... 34
3.4 The Extent of ER-Retention After Ribosome Dissociation Differs Among mRNA
Species ............................................................................................................................................................... 38
3.5 ALPP and CALR mRNAs Partially Target to the ER Independently of Translation ..... 47
3.6 Identification of Putative mRNA Receptors on the ER ........................................................... 49
3.7 Over-Expression of p180 Enhances the Ribosome- Independent Association of t-ftz
mRNA with the ER ........................................................................................................................................ 56
3.9 The Lysine-Rich Region of p180 Associates Directly with RNA In vitro ......................... 66
3.10 p180 Is Required for the Efficient Association of mRNA to the ER ................................ 67
3.11 p180 Is Required for the Translation- and Ribosome-Independent Maintenance of
ALPP and CALR mRNA at the ER. ............................................................................................................ 72
4.2 Efficient Translation-independent Maintenance of ALPP mRNA at the ER Requires
its Open Reading Frame ............................................................................................................................. 76
vi
4.3 The TMD Coding Region of ALPP mRNA Promotes the Translation-Independent
Maintenance of mRNA at the ER ............................................................................................................. 80
4.4 The Coding Potential of AP5 is not Required for ER-localization...................................... 82
4.5 The TMCR is Required for the Translational-Independent ER-Localization of ALPP86
4.6 AP5 Promotes the Efficient Targeting of mRNA to the ER Independently of
5.4 mRNAs encoding other exogenously expressed TA-proteins are mainly localized to
the cytoplasm .............................................................................................................................................. 105
5.5 The encoded TMD is not strictly required for the ER-localization of GFP-Sec61β
5.6 The initial targeting of GFP-Sec61β mRNAs to the ER is partially independent of
translation and ribosomes ..................................................................................................................... 108
5.7 p180 is not required for the localization of either GFP-Sec61β mRNA or its encoded
protein ............................................................................................................................................................ 110
5.8 TRC40 and BAT3 are not required for the localization of either GFP-Sec61β mRNA or
its encoded protein to the ER. ............................................................................................................... 113
5.9 GFP-Sec61β mRNA competes with other mRNAs for ribosome binding sites on the
ER ..................................................................................................................................................................... 115
3.7 Over-Expression of p180 Enhances the Ribosome- Independent
Association of t-ftz mRNA with the ER
The ER can be subdivided into morphologically distinct domains, such as the nuclear
envelope, perinuclear sheets, and peripheral tubules (18). Intriguingly, three of the membrane-
bound proteins from the ERMAP fraction-p180, kinectin, and CLIMP63-are abundant proteins
that localize to the perinuclear sheet portion of the ER, which is also enriched in translocon
components (156) and ribosomes (156,175). Interestingly, these three proteins diffuse into the
ER-tubules and nuclear envelope after puromycin or pactamycin treatment, indicating that their
enrichment in sheets is dependent on the integrity of polysomes and suggesting that they may
interact either with ribosomes or mRNA (156). In particular, p180 seems to be a suitable mRNA
receptor candidate. It has a very short luminal N-terminal segment followed by a single
transmembrane domain and a large C-terminal cytoplasmic region that is comprised of two basic
domains (a lysine-rich region followed by 54 tandem repeats of a basic decapeptide sequence)
and ends in a long coiled-coil domain. The highly charged domains are of particular interest as
they could potentially bind to the negatively charged phosphate backbone of RNAs. While p180
was initially identified as a ribosome receptor (176), more definitive experiments have shown
that the Sec61 translocon complex (57,58) and not p180 (177,178) is responsible for the majority
of ribosome binding activity present in ER-derived microsomes.
If p180 acts as a non-specific mRNA receptor, one would expect that the over-expression
of this protein would enhance the ribosome-independent ER-association of transcripts that
normally do not have this property. With this in mind we monitored the ER-association of t-ftz
mRNA in COS-7 cells that over-expressed green fluorescent protein (GFP)-tagged p180 (see
Figure 3.10A) in the presence and absence of HHT. As a control, we monitored the distribution
of t-ftz mRNA in cells expressing GFP-CLIMP63 and histone 1B-GFP (H1B-GFP). The
distribution of H1B-GFP, which binds to DNA in the nucleus, is not affected by extraction and
allowed us to identify co-expressing cells after digitonin- treatment. We observed that GFP-p180
over-expression promoted the ER-association of t-ftz mRNA in both control and HHT- treated
cells (Figure 3.10B-C). In contrast, over-expression of either GFP-CLIMP63 or H1B-GFP had
no effect (Figure 3.10B, D). Since the expression of GFP-p180 did not significantly affect the
cytoplasmic/nuclear distribution (Figure 3.11A) or the total level (Figure 3.11B) of t-ftz mRNA
in intact cells, we could rule out the possibility that the elevated level of ER-bound t-ftz was
caused by an upregulation of its nuclear export, production, or stability. Moreover, the level of
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nuclear t-ftz FISH signal did not significantly change, except for cells expressing H1B-GFP, and
this was likely due to the fact that these cells had a lower overall expression of t-ftz mRNA
(Figure 3.11B).
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Figure 3.10 Over-expression of p180 can enhance the ribosome-independent association of
t-ftz mRNA with the ER.
(A) COS-7 cells were transfected with plasmids containing vector alone (“mock”), GFP-p180 or
GFP-p180ΔLysΔRepeat. After 18-24 h cell lysates were collected, separated by SDS-PAGE and
immunoblotted for GFP, p180 or αtubulin. The position of molecular weight markers are
indicated on the left and proteins are labeled on the right. Note that a high molecular weight band
(denoted by an asterisk), which is positive for p180 and GFP, is detected in cells expressing
GFP-p180ΔLysΔRepeat. We suspect that this is an aggregate of the over-expressed protein. (B-E,
G-H) COS-7 cells were transfected with plasmids containing t-ftz gene alone (“mock”), or with
various GFP-tagged genes as indicated. The cells were allowed to express t-ftz mRNA and GFP-
tagged proteins for 18-24 h. Cells were then treated with either control media, or HHT for 30 min
to disrupt ribosomes, and then extracted, fixed, and stained for t-ftz mRNA using specific FISH
probes. (B, H) The fluorescence intensity of mRNA in the ER and nucleus in the micrographs
were quantified. Each bar represents the average and standard error of three independent
experiments, each consisting of the average integrated intensity of 30 cells over background. (C-
D, G) Each row represents a single field of HHT-treated cells (30 min) that was imaged for t-ftz
mRNA, and GFP. Cells co-expressing t-ftz mRNA and the GFP-tagged protein are denoted by
arrows, while cells that expressed only t-ftz are indicated by arrowheads. Scale bar = 20 μm.
Note that t-ftz mRNA remains associated to the ER in cells over-expressing GFP-p180 (C,
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arrows), but not GFP-CLIMP63 (D, arrows) or in cells expressing t-ftz alone (C-D and G,
arrowheads). Cells over-expressing GFP-p180ΔLysΔRepeat (G, arrows) show an intermediate
phenotype. (E) COS-7 cells that were transfected with plasmids containing t-ftz gene alone
(“mock”), or with various GFP-tagged genes, were lysed, separated by SDS-PAGE and
immunoblotted for p180, GFP, CLIMP63, translocon components (Sec61β and Trapα) or
αtubulin. (F) Domain architecture of the GFP-tagged p180 constructs. Both contain the CALR
SSCR to mediate proper protein translocation (purple), GFP (green), the p180 luminal region
which is 7 amino acids long and the p180 single pass transmembrane domain (TMD, orange).
The lysine-rich region (“Lys”, dark blue) and decapeptide repeat region (light blue) are present
only in the GFP-p180 construct. Both end with the p180 coiled-coil domain (red).
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Figure 3.11 Nuclear export of t-ftz mRNA remains unchanged in co-transfected cells, but
total t-ftz mRNA levels decrease in cells expressing H1B-GFP.
(A-B) COS-7 cells were transfected with either plasmids containing t-ftz alone or in combination
with plasmids containing GFP-p180, GFP-CLIMP63 or H1B-GFP. Cells were allowed to
express for 18-24 h, fixed and stained for t-ftz mRNA using specific FISH probes. Note that the
cells were not extracted prior to fixation. (A) The fraction of t-ftz mRNA in the cytoplasm and
nucleus in co-transfected cells. (B) The total level of t-ftz mRNA in the co-transfected cells,
normalized to cells expressing t-ftz alone. Each bar consists of the average and standard deviation
of 30-35 cells.
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Next, we investigated whether kinectin could act as a general mRNA receptor. In the
majority of cells, over-expression of GFP-kinectin did not promote a dramatic increase in the
level of ER-bound t-ftz mRNA (Figure 3.12A-B). However, we did observe a drop in nuclear t-
ftz mRNA compared to mock co-transfected cells. This was caused by a decrease in the total
level of t-ftz mRNA (Figure 3.12C) and not changes in cytoplasmic/nuclear distribution (Figure
3.12D). As the absolute level of ER-associated t-ftz mRNA after HHT-treatment did not change
(Figure 3.12B), despite the drop in its expression level (Figure 3.12C), we re-evaluated our data.
Upon closer inspection we found that in certain cells with high levels of GFP-kinectin, there was
an increase in the ribosome-independent ER-association of t-ftz (for example, see Figure 3.12E).
Indeed, in HHT-treated cells the level of ER-associated t-ftz correlated with the amount of co-
expressed GFP-kinectin, but not H1B-GFP (Figure 3.12F).
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Figure 3.12 GFP-kinectin over-expression slightly enhances the ER-association of t-ftz
mRNA after ribosome dissociation.
(A) COS-7 cells were transfected without (“mock”) or with plasmids containing GFP-p180 and
then lysed after 18-24 h. Cell lysates were separated by SDS-PAGE and immunobloted for
kinectin, GFP, αtubulin and translocon components (Sec61β and Trapα). (B-F) COS-7 cells were
transfected with plasmids containing t-ftz alone (“mock”) or in combination with GFP-kinectin
or H1B-GFP. After 18-24 h cells were either first extracted with digitonin and then fixed to
assess ER-association (B, E-F) or directly fixed to assess the total mRNA (C-D). After staining
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for t-ftz mRNA using specific FISH probes, cells were imaged. (B) The fluorescence intensity of
t-ftz mRNA in the ER and nucleus in extracted cells. (C) The total level of t-ftz mRNA in
unextracted cells. (D) The fraction of t-ftz mRNA in the cytoplasm and nucleus. (B-D) All data
points are normalized to “mock” (cells expression t-ftz alone). Each bar represents the average
and standard error of three independent experiments, each consisting of the average integrated
intensity of 30 cells over background. (E) A single field of HHT-treated cells (30 min) that was
imaged for t-ftz mRNA, and GFP-kinectin. Scale bar = 20 μm. Note that t-ftz mRNA remains
associated to the ER in cells with very high levels of GFP-kinectin (arrow), but not those with
low levels (arrowhead). (F) For each cell the total level of ER-associated ftz FISH signal
(normalized from the background (0), to the brightest cell (1); y-axis) was plotted against total
integrated GFP signal (normalized from the background (0), to the brightest cell (1); x-axis).
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We then examined whether over-expression of p180 affected the ER-association of bulk
mRNA. In cells expressing GFP-p180, there was almost a doubling in the amount of ER-
associated mRNA as compared to either H1B-GFP expressing, or untransfected cells (Figure
3.13). This was true for both untreated and HHT-treated cells. Although it is likely that a
substantial fraction of this enhanced ER-targeting was due to the recruitment of endogenous
transcripts, part of the observed increase was probably due to ER-bound GFP-p180 mRNA,
which is not present in the control transfected cells.
Figure 3.13 GFP-p180 over-expression enhances the ER-association of bulk poly(A)
mRNA.
COS-7 cells were transfected with either plasmids containing GFP-p180 or H1B-GFP and then
fixed after 18-24 h. Cells were then treated with either control medium, or HHT for 30 min to
disassemble ribosomes, and then extracted, fixed, and stained poly(A) mRNA using poly(dT)
FISH probes. (A) A single field of HHT-treated cells that was imaged for poly(A) mRNA and
GFP. Cells expressing GFP-p180 are denoted by arrows, while untransfected cells are indicated
by arrowheads. A cell with low GFP-p180 expression is denoted by an asterisk. Scale bar = 20
μm. The fluorescence intensity of mRNA in the ER was quantified (B). Each bar represents the
average and standard error of three independent experiments, each consisting of the average
integrated intensity of 50 cells over background.
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Since the expression of p180 has been shown to be important in up regulating secretion in
specialized secretory cells (179,180), our results could have been ascribed to an increase in
ribosome-anchoring proteins. However, cells over-expressing GFP-p180 and t-ftz did not have
altered levels of translocon components, such as Sec61β or Trapα, as seen by immunoblot
(Figure 3.10E). Over-expression of kinectin also had no effect on the levels of Sec61β or Trapα
(Figure 3.12A).
In order to determine whether the lysine-rich region and basic repeats were required for
ER-anchoring of mRNA, we over-expressed a version of GFP-p180 that lacks both these
domains (GFP-p180LysRepeat; Figure 3.10F) and monitored t-ftz distribution. Cells that
over-expressed this construct retained about half as much t-ftz on the ER after HHT treatment as
compared to cells over-expressing p180 (Figure 3.10F-G, see Figure 3.10A to compare the
expression levels of the two constructs). Notably this level of residual ER-associated t-ftz was
above control HHT-treated cells, indicating that GFP-p180-ΔLysΔRepeat still had some activity.
Interestingly, in the absence of translation inhibitors, cells expressing this construct had elevated
levels of ER-associated t-ftz mRNA (Figure 3.10H). This increase was not due to changes in
either the nuclear/cytoplasmic distribution or total levels of t-ftz mRNA in cells expressing GFP-
p180-ΔLysΔRepeat (Figure 3.11). Thus, it is likely that p180 has the ability to enhance the
translation-dependent association of t-ftz mRNA with the ER and that this activity does not
require the lysine-rich region or the basic repeats.
From these results we conclude that the over-expression of p180 promotes the ribosome-
and translation-independent association of mRNAs with the ER. Moreover, our data suggest that
this activity is mediated in part by the basic domains found in the cytoplasmic region of p180. In
addition, our results indicate that p180 stimulates the recruitment of mRNAs to the ER even in
the presence of translating ribosomes, however the basic regions are dispensable for this second
activity. In addition, it is likely that kinectin may have some weak ability to anchor mRNAs to
the ER independently of ribosomes.
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3.9 The Lysine-Rich Region of p180 Associates Directly with RNA In vitro
Next we investigated whether the basic cytoplasmic domains of p180 could associate
directly with RNA in vitro. In support of this idea we found that a bacterially expressed p180
lysine-rich region, fused to glutathione S-transferase (GST-p180-Lys; Figure 3.14A), could form
a complex with a 32
P-labeled RNA derived from the human insulin SSCR (Figure 3.14B). In
contrast, no complex was formed between this RNA and a control protein, GST-Ran (Figure
3.14A-B). By varying the amount of protein in our binding assay, we estimate that the GST-
p180-Lys binds to RNA with an affinity of about 0.8 μM. Since this protein could form
complexes equally well with other RNAs, such as a fragment of the human β-globin transcript
(unpublished data), it is unlikely that this domain has specificity for any particular sequence. We
also tested a peptide containing three copies of the consensus p180 decapeptide repeats;
however, we did not observe any complex between this reagent and any of the tested RNAs
(unpublished data). This result suggests that the repeats are not critical for mRNA interaction,
although we could not rule out the possibility that the peptide, which is 30 amino acids in length
and predicted to be disordered, failed to adopt some particular confirmation that is required for
RNA-interaction.
Figure 3.14 The lysine-rich region of p180 directly associates with RNA in vitro.
(A) GST-Ran and GST-p180-Lys were expressed in bacteria, purified using glutathione
sepharose, resolved by SDS-PAGE on a 12% acrylamide gel, and stained with Coomasie blue.
The size of relevant molecular weight markers (MWM) are indicated on the left. (B) 32
P-labeled
insulin SSCR RNA was incubated alone or with either GST-Ran or GST-p180-Lys for 15 min at
room temperature and then separated on a 10% non-denaturing TBE gel. Radiolabeled RNA was
visualized on a phosphorimager.
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3.10 p180 Is Required for the Efficient Association of mRNA to the ER
Next, we depleted p180, kinectin, or CLIMP63, by infecting U2OS cells with lentivirus
that deliver short hairpin RNA (shRNA) that are processed into small interfering RNA directed
against the human genes of interest. These treatments effectively depleted p180 and kinectin
(Figure 3.15A), but the level of CLIMP63 after shRNA knockdown was quite variable. In
addition depletion of CLIMP63 occasionally resulted in a decrease in kinectin levels (Figure
3.15A), however this was not consistent throughout all our experiments. Note that in these
preliminary experiments p180 was depleted with shRNA clone B9.
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Figure 3.15 p180 is required for the ER-association of mRNA.
(A–H) U2OS cells were infected with specific shRNAs against p180 (shRNA clones B9 and B10), kinectin or CLIMP63, or with
control lentivirus (“Cont”). (A-B) Cell lysates were separated by SDS-PAGE and immunoblotted for p180, CLIMP63, kinectin,
αtubulin, Trapα, and Sec61β. (C-E) Cells depleted of p180 (Clone B9; C, E) or kinectin (D), or infected with control lentivirus
(“cont”; C-E), were treated with control media (no drug, “ND”) or HHT for 30 min, then extracted with digitonin and stained for
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poly(A) mRNA using poly(dT) FISH probes. (C-D) For each cell the total level of ER-associated poly (A) FISH signal (normalized
from the background (0), to the brightest cell in the entire experiment (1), y-axis) was plotted against cell size (pixels squared, x-axis).
For each data set a regression line was plotted and the coefficient of determination (R2) was indicated. (E) The ratio of ER to nuclear
poly(A) fluorescence was quantified and normalized. Each bar represents the average and standard error of five independent
experiments, each consisting of the average of >30 cells. (F-H) Cells were depleted of p180 or kinectin with specific shRNAs, or
infected with control lentivirus, then transfected with plasmids containing either the ALPP (F–G) or CALR (H) gene. The cells were
allowed to express mRNA for 18-24 h, then treated with control media (no drug, “ND”) or HHT for 30 min, and then extracted with
digitonin. Cells were then fixed, stained for mRNA using specific FISH probes against the exogenous mRNA, and imaged. Nuclei are
outlined with blue dotted lines. Scale bar = 20 µm. (G-H) The fluorescence intensity on the ER and nucleus were quantified. Each bar
represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30
cells over background.
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Previously it was shown that the depletion of these three factors had no obvious effect
on ER-morphology except that CLIMP63 depletion decreased the average width of the ER
lumen (156). Moreover, p180 depletion did not significantly affect the level of translocon
components, such as Sec61β or Trapα (Figure 3.15B). We did observe, however, that the average
cell size increased after p180 depletion (Figure 3.16A). As a consequence, the total area occupied
by the ER and the nucleus also increased (Figure 3.16B). Since we also observed an increase in
bi-nucleate cells (unpublished data), it is possible that p180 is required to complete cytokinesis,
which would explain the increase in cell and nuclear sizes. We next determined whether p180
was required for the ER-association of bulk mRNA to the ER using poly(dT) FISH probes. To
control for changes in cell size, we imaged and quantified poly(A) FISH staining in extracted
cells and plotted the total fluorescence intensity in the ER versus the cell area for each cell.
When cells of a similar size were compared, we observed a decrease in the steady-state levels of
ER-associated mRNA after p180 depletion (Figure 3.15C). In contrast, kinectin depletion had no
effect on the level of ER-associated mRNA (Figure 3.15D). When p180 knockdown cells, which
already had a low level of ER-associated mRNA, were treated with HHT the amount of mRNA
on the ER only decreased slightly (Figure 3.15C). In contrast when control or kinectin-depleted
cells were treated with HHT, the amount of ER-associated mRNA dropped but was still higher
than what was seen in p180 knockdown cells with or without HHT treatment (Figure 3.15C-D).
In order to quantify the amount of ER-associated mRNA while controlling for changes in cell
size and variation in FISH signals between experiments, we normalized the integrated
fluorescence intensities of FISH signal in the ER to the nucleus for each cell. We found that
p180-depleted cells had significantly less ER-associated mRNA in comparison to control cells
(Figure 3.15E). Using this analysis, we found that HHT treatment reduced the amount of ER-
associated mRNA in both control and p180 knockdown cells, however even in the absence of
p180 and translation, there was still ER-associated transcripts (Figure 3.15E).
From these experiments we conclude that p180 promotes the efficient anchoring of bulk
mRNA to the ER, however as p180 depletion did not abolish the ribosome-independent ER-
association of mRNA, it is likely that other mRNA receptors exist.
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Figure 3.16 Depletion of p180 in U2OS cells increases cell size.
The area in square pixels of the whole cell (A) or the ER and the nucleus (B) were measured in
U2OS cells depleted of p180 with specific shRNAs or infected with control lentivirus and treated
with control media or HHT for 30 min prior to digitonin extraction. All values were normalized
to the size of either control cells (A) or ER (B). Each bar represents the average and standard
error of four independent experiments, each consisting of the average from >30 cells.
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3.11 p180 Is Required for the Translation- and Ribosome-Independent
Maintenance of ALPP and CALR mRNA at the ER.
We then tested the requirement of p180 for ER-association of specific transcripts by
analyzing the level of mRNA in the cytoplasm and the nucleus by FISH. p180 depletion reduced
the association of ALPP to the ER in both control and HHT-treated cells (Figure 3.15F-G). In
contrast to poly(A) staining, we did not observe an increase in nuclear ALPP in the knockdown
cells. We believe that this is due to the fact that the total amount of ALPP mRNA produced per
transfected cell did not change despite the increase in cell and nuclear size. Depletion of p180
with a second shRNA construct (clone B10, Figure 3.15B) also reduced the association of ALPP
to the ER in both control and HHT-treated cells (Figure 3.15G). In contrast, depletion of kinectin
had no effect on the level of ER-associated ALPP mRNA in either control or HHT- treated cells
(Figure 3.15-G). p180 depletion by either shRNA clone also reduced the ER-association of
CALR mRNA in both control and HHT-treated cells (Figure 3.15H).
From these experiments we concluded that the ribosome-independent anchoring of ALPP
and CALR mRNA to the ER requires p180.
3.12 Discussion
The work presented here provides, to our knowledge, the first molecular insight into how
a large fraction of ER-anchored transcripts are maintained on the surface of this organelle
independently of ribosomes in mammalian cells. Importantly, we demonstrate that the degree of
ribosome- and translation-independent targeting and maintenance at the ER varies greatly
between different transcripts. We then provide evidence that p180 acts as a general mRNA
receptor on the ER. Over-expression of a GFP-tagged version of this protein potentiates mRNA-
ER interaction, while its depletion reduces the amount of ER-associated mRNA. Finally we
demonstrate that p180 is required for the ribosome-independent anchoring of ALPP and CALR
mRNAs. Although p180 appears to be a metazoan-specific gene, recent findings have suggested
that mRNA may be anchored directly to membranes in prokaryotes (149), suggesting that the
ribosome-independent association of mRNAs to membrane-bound receptors is universally
conserved (150). Indeed our data suggest that other mRNA receptors for the ER exist in
mammalian cells. One potential candidate that we have yet to rule out is kinectin. Although its
depletion has little to no effect on the distribution of bulk poly(A) or ALPP mRNA (Figure 3.15
D,F,G), its over-expression promoted a small but detectable increase in the ribosome-
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independent association of t-ftz mRNA with the ER (Figure 3.12). Moreover, kinectin has a
cytoplasmic lysine-rich domain that resembles the RNA-binding region of p180. Future
experiments should determine the exact contribution of kinectin to this process.
Our results indicate that p180 and ribosome/translation dependent targeting mechanisms
act synergistically to enhance ER-anchoring of mRNAs (Figures 3.10B,H and 3.15E,G-H). In
agreement with our results, several groups have demonstrated that p180 expression promotes
secretion (179,180). Interestingly, the over-expression of p180 in budding yeast, which does not
express any endogenous p180-like proteins, leads to the proliferation of ER, the enhancement of
mRNA-ER association, and an increase in the half-life of ER-bound transcripts (181,182).
Furthermore, while ER- proliferation is stimulated by the over-expression of a version of p180
that lacks the basic domains, the enhanced mRNA-ER association requires these domains (182).
Although these results have been ascribed to the ability of p180 to directly recruit ribosomes, our
data support an alternative model where the basic domains of p180 associate directly to mRNA,
thus enhancing the partitioning of polysomes to the ER. It is also likely that p180 may have other
domains that mediate mRNA-ER association in mammalian cells. Indeed we found that the
expression of p180 lacking any basic regions (GFP-p180-LysRepeat) can promote ribosome-
dependent ER-anchoring of mRNA (Figure 3.10G-H). Taken together, our data suggest that the
coiled-coil domain may function primarily within the context of translation to enhance ER-
association. This result is in agreement with a recent study performed in collagen secreting cells
which demonstrated that p180 can promote the assembly of ER-bound polysomes, but that this
activity did not require its basic domains (183).
Importantly we demonstrate that p180 has a lysine-rich region that can directly bind to
RNA in vitro (Figure 3.14), likely through non-specific interactions with the mRNA backbone.
In light of this we predict that p180 acts in concert with proteins that recognize specific RNA
sequences to recruit particular mRNAs, such as ALPP and CALR, to the ER. Many candidate
proteins that could fulfill this function are likely found in the ERMAP fraction (Table 3.1).
Further studies will be required in order to determine whether these other ERMAP proteins play
a role in mediating specific interactions between mRNAs and the ER.
Intriguingly, our analysis also uncovered that the MSC, containing 10 tRNA synthetases,
and eEF1A1, which delivers charged tRNA to the ribosome, co-fractionates with ER-associated
mRNAs (Table 3.1). Recently it has been shown that the MSC not only co-fractionates with
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polysomes in a sucrose gradient, but also is distributed in a reticular pattern that is resistant to
cellular extraction with digitonin (184), suggesting that this complex associates predominantly
with ER-bound mRNA. It is possible that the MSC may mediate the direct delivery of charged
tRNAs to the ribosome (known as ‘‘tRNA channeling’’ (185)), and thus be responsible for the
enhanced rate of protein synthesis experienced by ER-targeted transcripts (186).
Finally, it is likely that mRNA receptors may restrict various transcripts to particular
subdomains of the ER. As mentioned previously, many asymmetrically localized mRNAs are
anchored by mRNA receptors that are present in particular ER-subdomains. This is best
illustrated in rice endosperm cells, where the transport and anchoring of specialized mRNAs to
specific ER-domains is dependent on an RNA binding protein that is homologous to
SND1/Tudor (21,187), a protein we identified in the ERMAP fraction (Table 3.1). Interestingly,
the differential distribution of ER-bound transcripts is also seen in mammalian cells. For
example, t-ftz, but not ALPP, appears to be excluded from the nuclear envelope (X. Cui and A.
Palazzo, unpublished observations). Moreover unlike translocon-associated proteins, which are
concentrated in ER-sheets (18,156), poly(A) appears to be distributed more evenly across all of
the ER (for example, compare the distribution of Trap and poly(A) in Figure 3.2A), suggesting
that the association of certain mRNAs with ER-tubules is mediated by interactions with some
additional unidentified RNA receptor(s). Ultimately, the restricted localization of certain mRNAs
may help to target newly synthesized proteins to distinct areas of the ER. This may be critical for
the proper localization of proteins with polarized distributions (19-22), especially for secretory
proteins that are exported at specific ER exit sites and are processed in specialized Golgi outposts
(188), which are present at peripheral cellular sites, such as in neuronal dendrites. The restricted
distribution of particular ER-bound transcripts may also be important to confine certain newly
synthesized ER-resident proteins which function in certain subdomains of this organelle, such as
the nuclear envelope (189). Again further analysis of RNA-binding proteins (particularly those
found in the ERMAP fraction), and their interacting RNA elements, will be required for a clearer
understanding of these processes.
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Chapter 4
Identification of a Region Within the Placental Alkaline Phosphatase mRNA that Mediates
p180 Dependent Targeting to the Endoplasmic Reticulum
This chapter is adapted from an article originally published as:
Cui, X. A, Zhang, Y, Hong, S.J. & Palazzo, A. F. Identification of a region within
the placental alkaline phosphatase mRNA that mediates p180 dependent targeting
to the endoplasmic reticulum. J. Biol. Chem 288(41): 29633-29641 (2013).
Acknowledgements:
Yangjing Zhang assisted with the image analysis for Figure 4.3 and constructed
Sec61-GFP and Sec22-GFP plasmid.
Seo Jung Hong contributed part of the data for Figure 4.2.
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4.1 Introduction
Thus far we determined that a subset of mRNA encoding secretory proteins can target
and be maintained on the ER independently of translation and this process is partially p180-
dependent. Next we wanted to identify the cis-acting RNA element(s) that promote(s) ER-
localization.
Previously, studies showed that transcripts such as human GRP94 (71), and CALR
(Chapter 3) mRNAs, which encode chaperones that reside in the lumen of the ER, and mRNA
encoding secreted human placental alkaline phosphatase (ALPP) protein can be maintained on
the ER independently of active ribosomes. In addition, mRNAs encoding certain cytosolic
proteins also co-fractionate with the ER (89). Importantly, both CALR and ALPP require p180
for their translation-independent maintenance at the ER (190). In contrast, certain engineered
reporter mRNAs, such as t-ftz, and natural transcripts, such as the INSL-3 and CYP8B1,
predominantly use the translation-dependent pathway for ER-localization (190,191). In light of
this, p180 likely interacts with additional RNA binding proteins, which have motif-
discriminating RNA-binding domains to provide specificity to select for mRNAs entering this
pathway.
In order to better understand this basic cellular process in mammalian cells, it is critical
that we identify RNA elements that promote p180-dependent ER-localization. To address this
question, we first delineated the region in ALPP mRNA that is responsible for the targeting of
this mRNA to the ER independently of translation. Then, we determined whether this process is
p180 dependent by depleting p180 in the cell using Lentiviral delivered shRNA.
4.2 Efficient Translation-independent Maintenance of ALPP mRNA at the ER
Requires its Open Reading Frame
In order to identify putative ER localizing RNA element(s), we created chimera
constructs between ALPP, which can be targeted and then maintained on the ER in a
translational independent manner, and t-ftz, which strictly requires translation for its localization
to the ER (Chapter 3). First we created two constructs that had UTRs from one gene and the ORF
of the other. AF1 contains the ORF of ALPP and the UTRs of t-ftz, while AF2 contains the
converse (Figure 4.1A). These constructs were transfected into COS-7 cells, which were then
allowed to express mRNA for 18-24 h. The cells were then treated with either control media,
puromycin (“Puro”), or homoharringtonine (“HHT”) for 30 min. The cells were then treated with
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digitonin to permeabilize the plasma membrane and extract any cytoplasmic mRNAs that are not
ER-bound, as previously described in Chapter 3. The cells were then fixed and AF1 mRNA was
detected by FISH using probes directed to the ALPP ORF, while AF2 was detected using probes
that hybridize to the ftz ORF.
Surprisingly, the majority of the AF1 mRNA was maintained on the ER after either
puromycin/EDTA, or HHT treatments. In contrast, the ER-association of AF2 mRNA was
sensitive to these treatments (Figure 4.2A-B). As an internal control we also monitored the
amount of nuclear mRNA, which is not affected by digitonin-permeabilization (Chapter 3), and
as expected this remained relatively unchanged among different treatment groups (Figure 4.2B).
As shown in Chapter 3, ALPP remained associated to the ER under all conditions, while t-ftz
required translation for ER-association. These results indicated that the ER-localizing cis-
element was present within the ORF of ALPP.
78
Figure 4.1 Schematic diagrams of constructs and U-content in ALPP and t-ftz.
A) Schematic of the chimeric constructs used in this study. All t-ftz sequences are depicted in
gray and ALPP sequences in white. B) Percent U content in ALPP, as analyzed using a moving
window of 50 nt, was plotted against the length of the construct. The signal sequence coding
region (SSCR), open reading frame (ORF), transmembrane domain coding region (TMCR), and
various fragments (AP1-5) are indicated. Note that the gray region represents AP5.
C) Percent U content in t-ftz, as analyzed using a moving window of 50 nt.
79
Figure 4.2 ALPP ORF can mediate the ribosome and translation independent mRNA localization on the ER in the chimera
construct.
COS-7 cells were transfected with plasmids containing either the AF1 or AF2 constructs and allowed to express mRNA for 18-24 h.
The cells were then treated with DMSO (“Ctrl”), puromycin (“Puro”), or HHT for 30 min, and then extracted with either digitonin
alone, or for puromycin-treated cells, with 20 mM EDTA. The cells were then fixed, stained for mRNA using specific FISH probes
(ALPP probe for AF1, ftz probe for AF2), and imaged. (A) Representative FISH images of cells expressing AF1 or AF2. (B)
Quantification of the fluorescence intensities of mRNA on the ER and nucleus. All data were normalized to the ER staining intensities
in the control treated group for each construct. Each bar represents the average and standard error of 3 independent experiments with
n>30 cells for each group. All scale bars = 20 m.
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4.3 The TMD Coding Region of ALPP mRNA Promotes the Translation-
Independent Maintenance of mRNA at the ER
The ALPP transcript is translated into a protein product that is translocated into the ER
and then anchored to the membrane by a carboxy-terminal TMD. This TMD is then cleaved and
the processed protein becomes covalently linked to a phosphatidyl-inositol glycan moiety, which
retains the protein at the membrane (192). Mature ALPP is then transported through the secretory
pathway to the surface of the cell.
To determine where the ER-localizing RNA element is located, we inserted 5 segments
of the ALPP ORF in between the signal sequence coding region (SSCR) and ORF of t-ftz (AP1,
AP2, AP3, AP4 and AP5) (Figure 4.1A-B). This allowed us to use the ftz FISH probe to detect
each of these chimeric mRNAs. Moreover, the presence of the t-ftz SSCR ensured that these
mRNAs would be properly exported from the nucleus to the cytoplasm (152). Plasmids
containing each construct were transfected into COS-7 cells. After 18-24 h, translation-
independent ER-association was assessed. We found that the AP5 fusion mRNA was maintained
on the ER independently of translation at a level similar to the full length ALPP (Figure 4.3A-B).
In HHT-treated cells, AP5 mRNA co-localized with Trapα, an ER marker, confirming that this
localization is ER specific (Figure 4.3C). Of the other chimeras, only AP2 showed a modest
increase in its localization on the ER in HHT treated cells in comparison to t-ftz, suggesting that
this fragment may have some limited localization capability. Note that a region of AP5 is also
found in AP4 (Figure 4.1B), however this later mRNA requires translation for efficient ER-
anchoring (Figure 4.3A-B).
From these experiments we conclude that the AP5 region of ALPP contains an ER-
localizing sequence. It is likely that the region responsible for this activity resides in the region
that is unique to AP5 (i.e., not found in AP4), in particular the TMD-coding region (TMCR).
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Figure 4.3 AP5 contains the
ER localizing RNA element.
COS-7 cells were transfected with
plasmid containing either full
length ALPP, t-ftz, AP1, AP2,
AP3, AP4 or AP5 and allowed to
express mRNA for 18-24 h. The
cells were then treated with
DMSO (“C” or “Ctrl”) or HHT
(“H”) for 30 min, and then
extracted with digitonin. Cells
were then fixed, stained for
mRNAs using specific FISH
probes (ftz probe was used to
detect AP1-5), and imaged. (A)
Quantification of the fluorescence
intensities of mRNA in the ER and
nucleus. All data were normalized
to the ER staining intensities in the
control treated group for each
construct. The results were
normalized to the ER staining
intensities in the control treated
group for each construct. Each bar
represents the average and
standard error of 3 independent
experiments with n>30 cells for
each group. (B) Examples of COS-
7 cells expressing either t-ftz, AP4
or AP5. (C) Example of a COS-7
cell expressing AP5 that has been
treated with HHT for 30min then
extracted with digitonin. The cell
was co-stained for AP5 using ftz
FISH probes and Trap, an ER
marker, by immunofluorescence.
All scale bars = 20 m.
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4.4 The Coding Potential of AP5 is not Required for ER-localization
To ensure that ER-localization of AP5 was not due to a low level of translation in HHT-
treated cells, we inserted a guanine between the 3rd
and 4th
nucleotide of the ORF of this mRNA,
creating frame-shifted AP5 (fs-AP5, Figure 4.1A). This mRNA encodes a polypeptide that does
not contain a signal sequence or TMD, as predicted by either SignalP (161) or TMHMM (160)
(data not shown). Furthermore, this peptide is mostly hydrophilic as measured by Kyte-Doolittle
Hydropathy plot (162) (Figure 4.4A), and is thus not a likely substrate for co-translational
translocation into the ER.
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Figure 4.4 Frame-shifted AP5 can still efficiently be maintained on the ER independently of ribosomes and translation. (A) Hydrophobicity (y-axis, left) of the polypeptides encoded by AP5 and fs-AP5 was plotted against the peptide length (x-axis,
bottom). The hydrophobicity was calculated with a moving window size of 21 amino acids. Note that AP5 encodes the t-ftz signal
sequence (residues 1-23 of the AP5 protein), followed by the C-terminal domain of ALPP (including the TMD of ALPP, residues 73-
90 of the AP5 protein), and then ends with the hydrophilic ftz protein. In contrast, fs-AP5 does not encode any hydrophobic region.
Also note that the frame shift mutation results in the creation of an early stop codon and thus a smaller encoded protein. For
comparison the U-content (x-axis, right) along AP5 (y-axis, top) was also plotted ((B-C) COS-7 cells were transfected with plasmid
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Figure 4.4 Frame-shifted AP5 can still efficiently be maintained on the ER independently of ribosomes and translation. (Continued) containing either t-ftz, AP5, fs-AP5 or H1B-GFP and allowed to express mRNA for 18-24 h. Cells were then fixed
directly (“Unextracted”) or digitonin extracted (“Extracted”) and stained with specific FISH probes (ftz probes were used to for AP5
and fs-AP5) to visualize mRNA distribution. (B) Representative examples. (C) Quantification of mRNA export and percent ER
localization. For each transcript, mRNA distribution between cytoplasm and nucleus was calculated in unextracted cells by comparing
the FISH staining intensity in each compartment to the total fluorescence within the cell. Percent ER localization of each transcript
was calculated by comparing mRNAs on the ER in the extracted cells with cytoplasmic mRNA content in unextracted cells. For each
group at least 40 cells were quantified, averaged and the ratio of these numbers was plotted. (D-E) Distribution of mRNAs in the
cytoplasmic and ER fractions. U2OS cells transfected with plasmid containing either t-ftz, fs-AP5 or M1-ftz (a cytosolic protein
containing a mRNA nuclear export promoting element M1) was fractionated into cytoplasm (“C”), ER (“ER”) and nuclear (“N”)
fractions. mRNA distribution between cytoplasmic and ER of each transcript was then determined via northern blot with [32
P]-labeled
ftz probe. (E) Various cell fractionation from transfected cells were analyzed by immunoblot using antibodies against either Trapα (a
resident ER protein), αtubulin (a cytoplasmic protein) and Aly (a nuclear protein). (F-G) COS-7 cells were transfected with plasmids
containing the t-ftz or fs-AP5 constructs and allowed to express mRNA for 18-24 h. The cells were then treated with DMSO (“Ctrl”) or
HHT for 30 min, and then extracted with digitonin. Cells were then fixed, stained for mRNA using FISH probes against ftz, and
imaged. (F) Representative images. (G) Quantification of the fluorescence intensities of mRNAs in the ER and nucleus. Each bar
represents the average and standard error of 3 independent experiments with n>30 cells for each group. (H) Example of a COS-7 cell
expressing fs-AP5 that has been treated with HHT for 30 min then extracted with digitonin. The cell was co-stained for fs-AP5 using
ftz FISH probes and Trap, an ER marker, by immunofluorescence. All scale bars = 20 μm.
85
Next, we expressed fs-AP5 in COS-7 cells and observed its distribution in normal and
digitonin-permeabilized cells in comparison to AP5 and t-ftz. We also monitored H1B-GFP
mRNA, which is not expected to be at the ER as it encodes a nuclear localized Histone 1B-Green
Fluorescent Protein fusion. In the majority of cells the distribution of t-ftz, AP5, fs-AP5 and H1B-
GFP mRNA was mostly diffuse in the cytoplasm indicating that a substantial fraction of these
transcripts were not ER-bound (Figure 4.4B). However, after digitonin-permeabilization,
significant levels of t-ftz, AP5, and fs-AP5, but not H1B-GFP mRNA remained in the cytoplasm
in a reticular pattern (Figure 4.4B, for quantification see 4.4C), that co-localized with Trapα (data
not shown). Indeed, we calculated that approximately one quarter of the cytoplasmic fs-AP5
mRNA is bound to the ER (Figure 4.4C). In contrast, less than 10% of the H1B-GFP mRNA was
ER-associated. We next confirmed this result by analyzing the distribution of various mRNAs in
different cell fractions. Transfected U2OS cells were separated into cytoplasm ER and nuclear
fractions as previously described (151,190), and the presence of RNA was determined by
northern blot. fs-AP5 and t-ftz were present at slightly higher levels in the ER than the
cytoplasmic fraction (Figure 4.4D). In contrast, M1-ftz mRNA, which encodes a cytosolic protein
and contains the mRNA nuclear export promoting element M1 (153), was present predominantly
in the cytoplasmic fraction (Figure 4.4D). To ensure that the fractions were not cross-
contaminated, we examined them for various markers. Note that the ER fraction was free of
cytosolic proteins, such as αtubulin, and nuclear factors, such as the RNA-binding protein Aly
(Figure 4.4E). It is likely that the discrepancy in the degree of ER-localization for fs-AP5
between Figures 4.4B and 4.4E was due to the fact that COS-7 cells tend to have higher levels of
expression than U2OS, and thus flood all of the mRNA-binding sites on the ER.
We then assessed whether the localization of fs-AP5 mRNA required translation. In
contrast to t-ftz, fs-AP5 remained ER-associated in COS-7 cells treated with HHT (Figure 4.4F-
G). This ER- localization in HHT-treated cells was confirmed by the co-localization of the
mRNA with the ER marker Trapα (Figure 4.4H).
These results confirm that AP5 contains an RNA element that can anchor transcripts on
the ER independently of translation.
86
4.5 The TMCR is Required for the Translational-Independent ER-
Localization of ALPP
We next deleted the last 90 nucleotides of the ORF from the full length ALPP. This
construct, ALPP-∆TMD, lacks the TMCR. Although this mRNA localized to the ER in COS-7
cells, we found that it was not retained there after HHT-treatment, especially when compared to
ALPP (Figure 4.5A-B). These observations indicate that the ER-retention element likely resides
in the TMCR.
87
Figure 4.5 The TMCD is required for the translational-independent ER-localization of
ALPP.
COS-7 cells were transfected with plasmid containing either ALPP or ALPP- ΔTMD and allowed
to express mRNA for 18-24 h. The cells were then treated with DMSO (“Ctrl”) or HHT for 30
min, and then extracted with digitonin. The cells were then fixed, stained for mRNA using
specific FISH probe against ALPP ORF. (A) Representative examples. (B) Quantification of the
fluorescence intensities of mRNAs in the ER and nucleus. Each bar represents the average and
standard error of 3 independent experiments with n>30 cells for each group. All scale bars = 20
m.
88
4.6 AP5 Promotes the Efficient Targeting of mRNA to the ER Independently
of Translation
Thus far our data indicates that once localized to the surface of the ER, AP5 is retained
there even after ribosomes have dissociated from the transcript. It is however possible that the
initial targeting of this mRNA to the ER is still dependent on translation, with the TMCR
mediating its subsequent ER- retention independently of translation. To test whether this region
can promote the initial targeting to the ER independently of translation, we first treated COS-7
cells with HHT for 15 min to halt all translation, then microinjected plasmids and monitored the
localization of the newly synthesized transcripts. In principle, an mRNA should be free of active
ribosomes throughout its entire lifetime under these conditions. We found that newly synthesized
AP5 was targeted to the ER in both control treated and in cells pre-treated with HHT (Figure
4.6A-B). The targeting of AP5 was less efficient than the full length ALPP, but enhanced when
compared to t-ftz (Figure 4.6B). These results indicate that AP5 can both target to, and be
retained on the ER independently of translation.
89
Figure 4.6 The initial ER-targeting of AP5 occurs independently of translation and
ribosomes.
(A-B) COS-7 cells were pretreated with DMSO (“Ctrl”) or HHT for 15 min, then microinjected
with plasmids containing either the ALPP, t-ftz or AP5. These plasmids were microinjected with
Alexa488-conjuaged 70KDa dextran, which marks injected cells and can be seen in the insets
(A). Cells were allowed to express mRNAs for 2 h in the presence of DMSO or HHT, then
extracted with digitonin, fixed and stained with specific FISH probes, and imaged. (A)
Representative examples. (B) Quantification of the fluorescence intensities of mRNAs in the ER
and nucleus. Each bar represents the average and standard error of 3 independent experiments
with n>30 cells for each group. All scale bars = 20 m.
90
4.7 p180 is Required for the Efficient Targeting of ALPP mRNA to the ER
Previously, we showed that p180 acts as an mRNA receptor that anchors several different
transcripts, such as ALPP, on the surface of the ER (Chapter 3). To test whether p180 was
required for the initial ER-targeting of this mRNA, we microinjected plasmids containing ALPP
into p180-depleted U2OS cells (Figure 4.7B). We observed that in the absence of p180, the
ability of the newly synthesized ALPP mRNA to target to the ER decreased in both control and
HHT-treated cells (Figure 4.7A, C). In contrast, the ER-targeting of t-ftz was strictly dependent
on active translation and was not affected by p180-depletion. These data indicate that p180 is
required not only for ER-anchoring, but also for the efficient ER-targeting of a subset of mRNA,
such as the ALPP transcript.
The fact that we can observe a p180-dependence in targeting even in the presence of
active translation, suggests that p180 also either works in parallel and/or directly enhances SRP-
dependent processes. This first model is supported by the fact that p180 is required for enhanced
ER-targeting even when translation is inhibited. The second possibility is supported by
observations that p180 may directly contact ribosomes (183) and/or the translocon (193).
Nevertheless our results strongly indicate that p180 is involved in both the initial targeting and
also the anchoring of ALPP on the surface of the ER.
91
Figure 4.7 p180 is required for
the initial ER-targeting of ALPP.
(A-C) U2OS cells were infected
with Lentivirus carrying control
shRNA (“Ctrl shRNA”) or shRNA
clone B10 against p180. The control
and p180-depleted cells were
pretreated with DMSO (“Ctrl”) or
HHT for 15 min, then microinjected
with plasmids containing either the
ALPP or t-ftz and allowed to
express mRNAs for 2 h in the
presence of DMSO or HHT. The
cells were then extracted with
digitonin, fixed and stained with
specific FISH probes, and imaged.
(A) Representative examples. (B)
Cell lysate was collected on the day
of injection and the level of
depletion was assessed by
immunoblotting against p180 and
tubulin. (C) Quantification of the
fluorescence intensities of mRNAs
in the ER and nucleus. Each bar
represents the average and standard
error of 3 independent experiments
with n>30 cells for each group. All
scale bars = 20 m.
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4.8 ER-maintenance of AP5 mRNA Requires p180
To examine whether TMCR-mediated ER localization is p180 dependent, we examined
the distribution of AP5 in U2OS cells depleted of p180 (Figure 4.8B). Indeed, similar to full
length ALPP mRNA, AP5 showed a significant decrease in its ability to associate with ER in
p180-depleted cells (Figure 4.8A,C). This decrease was seen in both control and HHT-treated
cells. Again the ability of t-ftz to be maintained on the ER was strictly dependent on translation
and was not affected by the depletion of p180.
In conclusion, our results indicate that the AP5 region of ALPP, which includes a TMCR,
contains an RNA element that anchors this mRNA to the surface of the ER in a p180-dependent
manner.
93
Figure 4.8 p180 is required for the ER association of AP5.
(A-C) U2OS cells were infected with Lentivirus carrying control shRNA (“Ctrl shRNA”) or
shRNAs (clones B9 or B10) against p180. The control and p180-depleted cells were transfected
with plasmids containing either the ALPP, t-ftz or AP5 constructs and allowed to express mRNAs
for 18-24 h. The cells were then treated with either DMSO (“C” or “Ctrl”) or HHT for 30 min
(“H”), digitonin extracted, fixed and stained with specific FISH probes, and imaged. (A)
Representative examples. (B) Cell lysate was collected on the day of injection and the level of
depletion was assessed by immunoblotting against p180 and tubulin. (C) Quantification of the
fluorescence intensities of mRNAs in the ER and nucleus. Each bar represents the average and
standard error of 3 independent experiments with n>30 cells for each group. All scale bars = 20
m.
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4.9 Discussion
We have identified a region of ALPP that contains an RNA element which promotes ER-
localization. Furthermore, our data indicates that this element requires p180 for its activity.
Interestingly, this element maps to a region of ALPP that encodes a TMD. This finding suggests
that mammalian cells may have a propensity to recognize nucleotide features that are associated
with certain protein coding regions. These inherent biases would be maintained by natural
selection primarily to constraints imposed by the encoded polypeptide, however subsequently
these features could be further exaggerated to enhance activities that act at the nucleotide level.
For example, the amino acid composition of TMDs is enriched in hydrophobic residues such as
Leu, Ile, Met, Val, and Phe. Interestingly, all of these amino acids are encoded by codons that
have a uracil at their second position and are thus relatively U-rich (154). Indeed, it has been
noted that uracil content is a good predictor of whether any given region within an ORF encodes
a TMD or a signal sequence (150). As mentioned in Chapter 1, it has been shown that E. coli
also targets certain mRNAs to the plasma membrane by elements found within TMCRs that are
U-rich (149). These results may indicate that the propensity for TMCR to promote the
localization of mRNAs to sites of secretory protein production, independently of translation.
This simplistic model however does not explain all of our observations. When the U-
content of the ALPP transcript was analyzed, the TMCR contains a relatively high level of Uracil
when compared to other sections of the transcript, however one can clearly find regions within
the ORF and UTRs that have even higher levels (see Figure 4.1B). Furthermore, regions of the t-
ftz 3′UTR (present in both t-ftz and AP5, see Figures 4.1C and 4.4A) have levels of U-content
that exceed what is found in the TMCR of ALPP, yet t-ftz does not exhibit much translational-
independent ER targeting. We also recently reported that the CYP8B1 mRNA, which encodes a
membrane- bound protein, also requires translation for efficient ER-anchoring (see Chapter 3),
suggesting that not all TMCRs have this activity. Finally, CALR mRNA, which does not encode
any TMD, associates with the ER independently of translation in a p180-dependent manner
(Chapter 3). Thus it is clear that although U-richness in TMCRs may help contribute to this
activity, other features are also important. Work in budding yeast has identified other cis-
elements that are responsible for ER targeting. One example is Pmp1, which contains several UG
repeats in its 3′UTR that can promote ER-localization (194). Recently, other yeast mRNAs have
been shown to localize to the ER independently of translation in manners that depend on either
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their ORFs and/or UTRs (195). Thus it is likely that many different elements may target mRNAs
to the ER.
Another element found within ORFs of metazoan mRNAs encoding secretory proteins is
the SSCR. It promotes nuclear export (152) and translation (151) of mRNAs. Mutations within
the SSCR redirect mRNAs from the ER to stress granules (152), large cytoplasmic aggregates of
mRNAs with stalled translation initiation complexes (196). SSCRs also have unusual nucleotide
compositions. They are depleted of adenine, enriched in guanine, cytosine and uracil, and tend to
contain CUG repeats (152,153). SSCRs however do not appear to promote translational-
independent ER-targeting as exemplified by t-ftz and INSL-3, which despite having SSCRs with
high U-content, absolutely require translation for efficient targeting and anchoring to the ER
(Figure 4.3A, also see Chapter 3). It is still possible that while the SSCR is not sufficient, it may
still be required for translation-independent ER-targeting.
We have attempted to analyze the degree of conservation in the 90 nucleotides at the end
of the ALPP ORF; however, this analysis was hampered by the fact that mammals contain
numerous ALPP paralogs, many of which lack TMCRs. Despite this, we found that these regions
had features that are associated with SSCRs. In particular, they have CUG repeats, GC-rich
regions and low adenine-content. These observations raise the possibility that the number of
SSCR-like segments present in a given mRNA impacts the efficiency of the p180-dependent
pathway. Future study is needed to identify p180-associated mRNAs and through this approach
we hope to computationally determine which sequence features, motifs or secondary structures
correlate with p180-dependency for ER-anchoring.
It remains unclear how p180 promotes the specific anchoring of a subset of mRNAs to
the ER. p180 likely binds the RNA backbone through ionic interactions (Chapter 3) and therefore
is not likely to act selectively. In light of this, other RNA-binding proteins, which have classic
RNA recognition motifs, may act in conjunction with p180 to selectively retain certain mRNAs.
Candidates were previously identified in a mass spectrometry screen of proteins that co-purify
with ER-derived polysomes (Chapter 3). Ultimately the ALPP TMCR could be used to identify
these accessory proteins.
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Chapter 5
mRNA Encoding Sec61, a Tail-Anchored Protein, is Localized on the Endoplasmic
Reticulum
This chapter is adapted from an article that has been submitted to the Journal of Cell
Science:
Cui, X. A. & Palazzo, A. F. mRNA Encoding Sec61a Tail-Anchored Protein, is
Localized on the Endoplasmic Reticulum. J. Cell Science, Submitted (2015).
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5.1 Introduction
One major mechanism that directs proteins to their correct subcellular destination is the
localization of their mRNA (197,198). Likely the most widespread example of this is the
localization of mRNAs encoding membrane and secreted proteins to the surface of the ER in
eukaryotic cells. This localization facilitates the targeting of the encoded proteins to the secretory
pathway (199). ER-localization of mRNAs is partially determined by the recognition of the
encoded polypeptide by the SRP system. In previous chapters, we demonstrated that a subset of
mRNAs also utilize a parallel mechanism, which is translation independent and partially
dependent on p180 (Chapter 3). In the ALPP mRNA, the cis-acting RNA element that mediates
this process resides within the TMCR (Chapter 4). In this chapter, we will examine the
localization of mRNAs encoding TA-proteins.
Tail anchored proteins account for about 5% of membrane anchored proteins in the cell.
This group of proteins does not contain an N-terminal signal sequence; instead they are targeted
and anchored to the phospholipid bilayer by a single hydrophobic transmembrane domain close
to the COOH terminus. They are involved in many essential cellular processes. Some prominent
examples of this class of protein include members of SNARE proteins (Soluble NSF Attachment
Protein), which mediate vesicle transport; and Bcl2 family of proteins, which are regulators of
the cellular life-or-death switch.
It is currently believed that TA-proteins are synthesized by cytoplasmic (i.e. non ER-
bound or “free”) ribosomes. Upon the completion of translation these proteins are post-
translationally targeted to the ER membrane via a cascade of protein chaperones (Figure 1.1C).
In the mammalian system, as the translation of tail-anchored protein finishes, the protein exits
the ribosome and is recognized by the pre-targeting recognition complex consisting of Bat3-
TRC35-Ubl4A (97,100). This complex then hands the TA-protein to TRC40, a conserved
cytosolic chaperone that recognizes and delivers the TMD of TA-proteins to the ER
membrane(78,97-100). Recently, the ER membrane receptor for TRC40 was identified to be a
heteromultimer composed of CAML and WRB, which interacts with the incoming TRC40 and
mediates the insertion of TA-protein into the ER membrane (97,100,103). This model is largely
based on studies in yeast and in vitro reconstitution of the protein-targeting pathway using
mammalian cell fractions (78,96,97). Interestingly, it has never been demonstrated that TRC
components are required for proper TA-protein localization in intact mammalian cells. Moreover,
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many of the TRC/GET components are dispensable for cell viability despite the fact many of the
TA-protein substrates are essential for life. This discrepancy suggests that an alternative
biogenesis mechanism exists for this group of proteins.
Here we investigated the alternative mechanism for the biogenesis of TA-proteins
including Sec61β and Nesprin2. By studying both overexpressed and endogenous mRNA
transcripts, our study demonstrates that some mRNAs encoding the TA-proteins, such as Sec61β,
associate with the ER. In addition, the ER-association of Sec61β mRNA is not dependent on
TRC40, BAT3 or p180. Moreover, overexpression of Sec61β mRNA displaces other mRNAs
from the ER, including those that are anchored by translocon-bound ribosomes. This suggests
that some mRNAs encoding TA-proteins can access translocon-bound ribosomes on the surface
of the ER.
5.2 Sec61β mRNA is partially localized on the ER
It is currently believed that mRNAs encoding TA-proteins are first translated by free
ribosomes, and that the encoded polypeptide is later post-translationally targeted to the ER via
the GET/TRC pathway (105,110,200).
To assess the distribution of endogenous mRNA in human cells, we stained U2OS cells
with a panel of fluorescent in situ hybridization (FISH) probes. By simultaneously staining with
many probes, one can efficiently visualize individual mRNA molecule (201), as can be seen in
Figure 1. To determine whether these RNAs were tethered to the ER we repeated the experiment
in cells that were treated with digitonin, which permeabilizes the plasma membrane and thus
extracts the cytoplasm and removes any molecule that is not ER-associated (89,190,191). By
comparing the number of puncta in non-extracted versus extracted cells, we can determine the
percentage of mRNAs are anchored to the ER.
First we examined the localization of Sec61β mRNA, which encodes a TA-protein.
Sec61 is a component of the translocon, the major protein-conducting channel in the ER, and
has been widely used as a model GET/TRC pathway substrate (110). Surprisingly we found that
about 30% of the endogenous Sec61β mRNA was resistant to digitonin extraction (Figure 5.1A-
B). To test whether the localization of Sec61β mRNA was translation dependent, we examined
the mRNA localization in cells treated with either homoharringtonine (HHT) or puromycin and
extracted with EDTA (Puro+EDTA), two treatments that effectively dissociate ribosomes from
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mRNA (190). To our surprise, most of the ER-localized mRNA was unaffected by these
treatments.
Figure 1. Endogenous Sec61β
and Nesprin2 mRNA
associates with the ER
membrane.
U2OS cells were either: fixed
(“Unextracted”); first extracted
with digitonin and then fixed
(“Extracted”); or pre-treated
with Puromycin (Puro) or
Homoharringtonine (HHT) for
30 min, extracted with
digitonin in the presence or
absence of EDTA and then
fixed. Cells were stained with a
pool of FISH probes to
visualize individual
endogenous human
Sec61βNesprin2 or GAPDH
mRNA molecules. Each cell
was visualized by phase
microscopy to determine the
cell contours. mRNA foci were
identified using NIS-element
“Spot Detection” function (see
Methods section). Shown in (A)
are the mRNA FISH signals
overlaid with the contours of
the cells and nuclei and the
detected foci highlighted by the
spot detection function. (B)
The number of cytoplasmic (i.e.
non-nuclear) foci were
determined for each condition.
Each bar is the average and
standard error of 30 cells.
Next we monitored the localization of Nesprin2 mRNA, which encodes a giant TA-
protein (796 kDa) that is present on the outer nuclear envelope and is involved in nuclear
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positioning (202). After extraction about two thirds of the foci remained, indicating that some of
this mRNA was anchored to the ER (Figure 5.1A-B). To ensure that the FISH signal was specific,
we also probed cells that were depleted of their endogenous Nesprin2 mRNA using RNAi.
Indeed, RNAi-treated cells lost 90% of their signal (Figure 5.2), indicating that our Nesprin2
probes detected the intended target. Like Sec61β, Nesprin2 mRNA largely remained ER-
associated in cells treated with HHT and puromycin/EDTA. Thus Nesprin2, like Sec61β, can
associate with the ER-membrane and that this activity is mostly independent of translation.
To determine whether partial ER-association was a general phenomenon for all mRNAs,
we next investigated the localization of an mRNA encoding a cytosolic protein, glyceraldehyde
3-phosphate dehydrogenase (GAPDH). We could reproducibly find 15% of the GAPDH puncta
in digitonin-extracted cells (Figure 5.1A-B). However, in contrast to what we had seen for
Sec61β and Nesprin2, most of the GAPDH mRNAs were extracted in cells treated with either
HHT or puromycin/EDTA (Figure 5.1), suggesting that the small amount of ER-association was
mediated by translating ribosomes.
Thus we concluded that at least two endogenous mRNAs that encode TA-anchored
proteins are also ER-associated, and this was mostly mediated by contacts that did not involve
the ribosome.
Figure 5.2. Endogenous Nesprin2 mRNA
localization in U2OS cells.
U2OS cells were treated with control medium
or HHT for 30 min then either fixed directly
or first digitonin-extracted, stained with
specific pools of FISH probes against
endogenous human Nesprin2, and imaged.
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5.3 The ORF of Sec61 mRNA is required to anchor to the ER independently
of translation
We next wanted to identify the region of Sec61 mRNA responsible for its ER-anchorage.
We followed a strategy that we had previously used to identify regions in the placental alkaline
phosphatase (ALPP) mRNA that promoted ER-anchorage (203). We fused different regions of
Sec61 to t-ftz (Figure 5.3A), an artificial mRNA that encodes a secretory protein and requires
translation for ER-association (190). These constructs were expressed in COS7 cells. After 18-24
hrs, cells were either treated with control or HHT for 30min to disrupt ribosomes, then extracted
to remove non-ER associated mRNAs, and then stained by FISH. To our surprise, versions of t-
ftz containing either the 5’UTR (5’UTR-t-ftz) or 3’UTR (3’UTR-t-ftz) of Sec61 did not remain
anchored to the ER after HHT-treatment, resembling the original t-ftz mRNA (Figure 5.3B, for a
quantification of the fluorescence intensity, see 5.3C). In contrast, a version of t-ftz fused to the
Sec61 ORF (t-ftz-ORF) remained ER-associated after HHT-treatment (Figure 5.3B). In fact
quantification of the FISH intensities revealed that the level of ER-association did not
significantly change between control and HHT-treated cells (Figure 5.3C).
To further validate these findings we examined the distribution of GFP-Sec61, a
construct that contains the ORF of the human Sec61 gene (Figure 5.3A). In unextracted COS7
cells the mRNA had a noticeable reticular-like distribution, suggesting that a large fraction of
this mRNA may be localized to the ER (Figure 5.3D). In digitonin-treated cells, a large portion
of the GFP-Sec61β mRNA was resistant to extraction (Figure 5.3D). In these cells GFP-Sec61β
mRNA co-localized with its translated product, GFP-Sec61β protein (Figure 5.3E), which is a
well-established marker of the ER (204). In contrast, H1B-GFP mRNA, which encodes a nuclear
histone protein, was mostly extracted by digitonin treatment (Figure 5.3D). When the FISH
fluorescence levels in extracted and unextracted cells were compared, we observed that 60% of
the GFP-Sec61β mRNA was resistant to extraction (Figure 5.3F). This is comparable to what we
previously observed for other over-expressed mRNAs encoding secreted and membrane-bound
proteins (190,191). In contrast, only about 10% of H1B-GFP mRNA was resistant to digitonin
extraction (Figure 5.3F), which is also in line with our previous observations (190).
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Figure 5.3. Overexpressed GFP-Sec61β mRNA is associated with the ER membrane.
(A) Schematic diagram of constructs. All t-ftz sequences are shown in white, Sec61β sequences
are shown in grey and EGFP sequences are shown as checked boxes. (B-C) Chimera plasmids
containing either the Sec61β 5’UTR, 3’UTR or the ORF fused to t-ftz were transfected into
COS7 cells. 18-24 hrs post-transfection, cells were treated with either control or HHT, followed
by digitonin extraction to remove cytoplasmic contents. Cells were fixed, stained using FISH
probes against ftz, and imaged. (D-F) Plasmids encoding GFP-Sec61β or H1B-GFP were
transfected into U2OS cells. 18-24 hrs post transfection, cells were either fixed directly
(“Unextracted”) or after digitonin extraction (“Extracted”). GFP-Sec61β or H1B-GFP mRNAs
were stained with FISH probes against the GFP-coding sequence and visualized. mRNAs in
unextracted and digitonin-extracted cells are shown in (D). Note that GFP-Sec61β, but not H1B-
GFP mRNA, is resistant to digitonin extraction and exhibits a reticular staining pattern. (E)
Distribution of GFP-Sec61β protein and mRNA in a digitonin-extracted U2OS cell. Both images
are from a single field of view. Note the extensive colocalization of the mRNA with its encoded
protein, which is localized to the ER. (F) Quantification of GFP-Sec61β and H1B-GFP mRNA
cytoplasmic intensity signals. The ratio of fluorescence in the cytoplasms of extracted versus
unextracted cells was determined. Each bar represents the average and standard error of 3
independent experiments, each containing at least 30 cells. All scale bars = 20µm.
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Next we assessed whether ER-association of GFP-Sec61β mRNA required translation.
Neither puromycin/EDTA nor HHT treatment disrupted the ER-association of GFP-Sec61β
mRNA in COS7 cells, as assessed by digitonin extraction (Figure 5.4A-B). HHT-treatment only
slightly decreased the ER-localization of this mRNA in U2OS cells (Figure 5.4C-D). To control
for differences in mRNA expression and staining efficiency, we also measured the nuclear
fluorescence, and this did not change under any of the tested conditions (Figure 5.4B-C). The
localization of GFP-Sec61β mRNA to the ER in HHT-treated U2OS cells was confirmed by
colocalization of the mRNA with the ER-marker Trapα (Figure 5.4E).
From these experiments we concluded that the ORF of Sec61β mRNA can promote ER-
association and that this activity is largely independent of ribosome-association and active
translation.
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Figure 5.4. ER-association of overexpressed GFP-Sec61β mRNA is partially independent of
translation.
(A-B) COS7 and U2OS (C-D) cells were transfected with plasmid encoding GFP-Sec61β and
allowed to express mRNA for 18-24 hrs. Cells were then treated with DMSO (“Ctrl”),
puromycin (“Puro”) or homoharringtonin (“HHT”) for 30 min, and then extracted with digitonin
with or without EDTA. Cells were then fixed, stained for mRNAs using a specific FISH probe
against the GFP-coding sequence. Cells were imaged (A,D), and the fluorescent intensities were
quantified (B,C). To control for changes in staining, nuclear fluorescent intensities were also
analyzed. Each bar represents the average and standard error of 3 independent experiments, with
each experiment consisting of at least 30 cells. (E) U2OS cells expressing GFP-Sec61β were
treated with HHT and then digitonin-extracted. Cells were then stained for the GFP-Sec61β
mRNA, and immunostained with the ER marker Trapα. Images in (E) are from a single field of
view including a color overlay showing the GFP-Sec61β mRNA in red and Trapα in green. All
scale bars = 20µm.
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5.4 mRNAs encoding other exogenously expressed TA-proteins are mainly
localized to the cytoplasm
To determine whether the results obtained with GFP-Sec61β mRNA can be generalized
to other mRNAs encoding TA-proteins, we examined the localization of other overexpressed
GFP-fusion transcripts. In particular we analyzed the distribution of mRNAs containing ORFs
that encode TA-proteins destined to the ER (Sec22β and Sec61γ), peroxisome (Pex26) or
mitochondria (FIS1). Previously, it has been shown that newly synthesized Pex26 protein is
targeted to the peroxisome via Pex19 and thus is independent of the TRC40 dependent pathway
(121). For TA-proteins destined for the mitochondria, they are thought to be recognized by a pre-
targeting complex which then prevents their sorting to the ER and instead diverts these to the
mitochondrial outer membrane (98). This sorting process is thought to be dictated by the relative
hydrophobicity of the TMD and the presence of charged residues in the vicinity of the TMD
(98,119,205).
As expected, GFP-Sec22β and GFP-Sec61γ proteins were targeted to the ER in COS7
cells (data not shown). Likewise, GFP-FIS1 and GFP-Pex26 proteins were targeted, as expected,
to the mitochondria (Figure 5.5) and peroxisomes (data not shown) respectively. However,
unlike GFP-Sec61β, all of the other tested mRNAs were efficiently removed by digitonin-
extraction (Figure 5.6A, compare “Cyto/ER” levels in unextracted to extracted cells), similar to
what was seen for mRNAs encoding non-secretory proteins (H1B-GFP; Figure 5.6A).
We next explored the idea of whether the targeting of a mitochondrial TA-protein to the
ER would also increase the amount of ER-targeting of its mRNA. When we increased the
hydrophobicity of the TMD of FIS1 (FIS1-5L, Figure 5.5B), the protein was successfully
rerouted to the ER (Figure 5.5A). However, the mRNA of GFP-FIS1-5L was still sensitive to
extraction and did not localize to the ER (Figure 5.6A).
From these experiments we concluded that ER-targeting of the protein product is not
sufficient for the ER-localization of an mRNA.
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Figure 5.5. Incorporation of leucines in the TMD of FIS1 reroutes the protein from the
mitochondria to the ER.
(A) COS7 cells were transfected with plasmid encoding GFP-FIS1 or GFP-FIS1-5L, which
contains 5 leucine mutations (see the sequence in panel B). Cells were either directly fixed
(“unextracted’) or first extracted with digitonin then fixed. The fixed cells were stained with
DAPI and immuostained for either ATP5A (a mitochondrial marker), or Trapα (an ER marker).
Each row represents a single field of view including overlays of GFP-FIS1/FIS1-5L (green),
ATP5A/Trap (red) and DAPI (blue) Scale bar = 20m.
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Figure 5.6. The coding potential of GFP-Sec61β is not required for its localization to the
ER.
(A) COS7 cells were transfected with plasmid encoding various GFP tagged TA-proteins and
allowed to express for 18-24 hrs. The cells were treated with control medium or HHT for 30 min,
then either directly fixed or extracted with digitonin and then fixed. Cells were stained for
mRNAs using specific FISH probe against the GFP coding sequence, imaged and quantified.
Fluorescent intensities in the cytoplasm and nucleus were quantified. All results were normalized
to the cytoplasmic staining intensity in the unextracted cells. Each bar represents the average and
standard error of 3 independent experiments, each consisting of at least 30 cells. (B)
Hydrophobicity (y-axis, left) of the polypeptides encoded by GFP-Sec61β and GFP-fs-Sec61β
was plotted against the peptide length (x-axis, bottom). Kyte-Doolittle Hydropathy values were
computed with ProtScale (http://web.expasy.org/protscale/), using a moving window size of 21
amino acids. Note the high hydrophobicity of the TMD region of GFP-Sec61β that is lost in
GFP-fs-Sec61β. (C) COS7 cells were transfected with plasmid encoding GFP-fs-Sec61β and
allowed to express mRNA for 18-24 hrs. Cells were then treated with control medium or HHT
for 30 min, and then either fixed (“Unextracted”) or extracted with digitonin and then fixed
(“Extracted’). Cells were stained for mRNAs using a specific FISH probe against the GFP-
coding sequence, and for DNA using DAPI. Each row represents a single field of view imaged
for GFP-fs-Sec61β mRNA, GFP protein and DAPI. (D) Quantification of the cytoplasmic (in
unextracted cells), ER (in extracted cells) and nuclear fluorescence intensities of GFP-fs-Sec61β
mRNA. Each bar represents the average and standard error of 30 cells. All scale bars = 20µm.