RNA Binding Protein, Ybx2, Regulates RNA Stability …...RNA binding protein, Ybx2, regulates RNA stability during cold-induced brown fat activation Dan Xu1,2*, Shaohai Xu3, Aung Maung

RNA binding protein, Ybx2, regulates RNA stability during cold-induced brown fat

activation

Dan Xu1,2*, Shaohai Xu3, Aung Maung Maung Kyaw2, Yen Ching Lim1, Sook Yoong Chia2,

Diana Teh Chee Siang2, Juan R. Alvarez-Dominguez5, Peng Chen3, Melvin Khee-Shing

Leow6,7,8, Lei Sun2,4*

1School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou,

Zhejiang 325035, China

2Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 8 College

Road, Singapore 169857, Singapore

3Division of Bioengineering, Nanyang Technological University, 70 Nanyang Drive,

Singapore 637457, Singapore 4Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673,

Singapore 5Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard

University, 7 Divinity Avenue, Cambridge, MA 02138, USA 6Clinical Nutrition Research Centre, Singapore Institute for Clinical Sciences, Agency for

Science, Technology and Research (A*STAR), Singapore, Republic of Singapore. 7Department of Endocrinology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng,

Singapore 308433, Singapore 8Office of Clinical Sciences, Duke-NUS Medical School, 8 College Road, Singapore 169857,

Singapore

*Correspondence: [email protected] (D.X.); [email protected] (L.S.)

Page 1 of 51 Diabetes

Diabetes Publish Ahead of Print, published online September 29, 2017

Abstract

Recent years have seen an upsurge of interest on brown adipose tissue (BAT) to combat the

epidemic of obesity and diabetes. How its development and activation are regulated at the

post-transcriptional level, however, has yet to be fully understood. RNA binding proteins

(RBPs) lie in the center of post-transcriptional regulation. To systemically study the role of

RBPs in BAT, we profiled >400 RBPs in different adipose depots and identified Y-box

binding protein 2 (Ybx2) as a novel regulator in BAT activation. Knockdown of Ybx2 blocks

brown adipogenesis, while its overexpression promotes BAT marker expression in brown

and white adipocytes. Ybx2 knockout mice could form BAT but failed to express a full

thermogenic program. Integrative analysis of RNA-seq and RNA-immunoprecipitation study

revealed a set of Ybx2’s mRNA targets, including Pgc1α, that were destabilized by Ybx2

depletion during cold-induced activation. Thus, Ybx2 is a novel regulator that controls BAT

activation by regulating mRNA stability.

Page 2 of 51Diabetes

INTRODUCTION

Obesity has reached an epidemic scale in many countries, resulting in a steep escalation in

health care expenditure and a growing burden of chronic obesity-related morbidities(1). An

attractive approach to improve metabolic health is to augment the mass and activity of brown

adipose tissue (BAT)(2-7). There are at least two types of thermogenic adipocytes in

mammals, namely, classical brown adipocytes and inducible/beige adipocytes. Classical

BAT is located as a discernible depot in the interscapular region in small mammals and

human infants. Beige/inducible adipocytes exist in defined anatomical white adipose tissue

(WAT) depots, particularly in subcutaneous WAT, and express a gene program more like

WAT at thermoneutrality. In response to prolonged cold exposure, chronic treatment of β-

adrenergic receptor agonist, or intensive exercise, the number of beige adipocytes

dramatically increases, accompanied by enhanced Ucp1 levels and mitochondria biogenesis,

a process known as “browning” (2; 5; 6).

Understanding the detailed mechanisms underlying BAT differentiation and function is an

area of immense research interest. A vast array of factors has been identified that regulate

BAT development and activity by acting at the transcriptional level(6-16). How these

processes are regulated at the post-transcriptional level, however, has yet to be fully

understood. RNA binding proteins (RBPs) comprise a large and diverse group(17; 18) that

lie at the center of posttranscriptional regulation by governing the fate of mRNA transcripts

from biogenesis, stabilization, translation to RNA decay. Several RBPs have been reported

to modulate adipocyte development and lipid metabolism. SFRS10 (splicing factor

arginine/serine-rich10) inhibits lipogenesis by controlling the alternative splicing of LPIN1, a

key regulator in lipid metabolism(19; 20). Sam68 (the Src-associated substrate during

mitosis of 68 kDa) is required for white adipose tissue (WAT) adipogenesis by regulating

mTOR alternative splicing(21). Knockout of KSRP (KH-type splicing regulatory protein)

promotes browning of WAT by reducing miR-150 expression(22). IGF2 mRNA binding

protein 2 (IGF2BP2) is a widely expressed RBP and a SNP in its intron is associated with

type 2 diabetes mellitus by GWAS studies(23). Knockout of IGF2BP2 results in resistance to

diet-induced obesity, largely due to an enhanced translational efficiency of Ucp1 and other

mitochondria mRNAs in the knockout BAT(24). Recently, paraspeckle component 1 (PSPC1)

was identified as an essential RBP for adipose differentiation in vitro and in vivo by

regulating the export of adipogenic RNA from nucleus to cytosol (25). Despite these

advances, our understanding of RBPs in adipocytes, particularly in brown adipocytes, is still

at its early stage and the functions of most RBPs remain unknown.

In this study, we systemically profiled 413 RBPs in different fat depots, during white fat

browning and brown adipogenesis, and identified 5 BAT-enriched RBPs. We demonstrated


the role of Ybx2 in development and activation of BAT in vitro and in vivo, which could be, at

least partially, explained by stabilizing mRNA.

METHODS

Animal Studies

Ybx2 heterozygous mice (NSA (CF-1) Background) were originally imported from Dr. Paula

Stein in University of Pennsylvania. C57BL6 mice were obtained from The Jackson

Laboratory and subsequently bred in house. All mice were maintained at the animal vivarium

at DUKE-NUS Medical School. For cold challenge experiments, animals were housed

individually in a 4oC chamber for 6 hours. The rectal body temperature was recorded with a

probe thermometer (Advance Technology) at a constant depth. All animal experimental

protocols were approved by the Singapore SingHealth Research Facilities Institutional

Animal Care and Use Committee.

Glucose tolerance test (GTT) and Insulin tolerance test (ITT) was performed as described

before (26) and EchoMRI was used to measure fat and lean mass. For in vivo insulin

signalling study, Ybx2 KO and WT mice were fasted for 6hr at RT or 4oC. Then the mice

were injected with insulin (1 U per kg body weight). After 5 min, mice were sacrificed and

BAT were collected. Lipolysis assay was performed as described before (26).

Cell culture

293T cells for retroviral packing were cultured in Dulbecco's modified Eagle medium

containing 10% fetal bovine serum (FBS) (HyClone™). Primary brown and white pre-

adipocytes were isolated from 3-4 week old C57BL6 mice. The procedure for pre-adipocytes

isolation, culture and differentiation and Oil Red O staining was described previously (26).

Human primary interscapular brown adipocytes were obtained from Zenbio Inc and cultured

and differentiated as previously described (27).

Retrovirus transduction

A MSCV based retroviral vector (MSCV-pgkGFP-U3-U6P-Bbs vector)(28) was used to

generate shRNAs to infect preadipocytes; XZ201 vector(29) was used to overexpress Ybx2

for gain-of-function studies. All the retroviruses were packaged in 293T cells with the pCL-

eco packaging vector and then used to transduce pre-adipocytes in the presence of 4 mg/ml

polybrene (Sigma), followed by induction of differentiation. FuGENE® 6 Transfection

Reagent (Promega) was used for plasmid transfection according to manufacturer’s

instruction.

RNA immunoprecipitation (RIP).

Primary brown and white adipocytes were infected with retroviral Ybx2 and differentiated for

4 days. RNA immunoprecipitation was performed using Magna RIP kit (Merck Millipore)


according to manufacturer’s instruction. RNA samples retrieved from Anti-Ybx2 (Abcam) and

IgG control with Magna RIP kit were used for RNA-seq.

RNA pull-down

RNA pull-down was performed according to our published protocol with a few modifications

(30). In this study, we used tissue lysate from mouse BAT instead of primary cell culture

prepared as described above. The tissue lysate was prepared as described in the RIP

section. The rest of the experiment followed our published protocol (30).

Extracellular Flux Analysis

Primary brown pre-adipocytes were seeded in an X-24 cell culture plate, infected by

retroviral constructs as indicated in the text, followed by induction of differentiation.

Differentiated cells were analyzed by Extracellular Flux Analyzer (Seahorse bioscience)

according to the manufacturer's instructions. Oxygen consumption rates were normalized by

protein concentration.

Animals were kept at 4oC for 6 hours before experiment. BAT and skeletal muscle

(Gastrocnemius) were harvested and minced with micro-mincer (Glen Mills Inc). The minced

tissue was kept in ice-chilled mitochondrial respiration media (MiR05) (EGTA 0.5mM,

MgCl2.6H2O 3mM, Lactobionic acid 60mM, Taurine 20mM, KH2PO4 10mM, HEPES 20mM,

D-Sucrose 110mM, BSA 1g/l). 2mg and 10mg tissue lysate, respectively, was immediately

loaded into Oroboros Respirometry together with substrates including Glutamate, Malate,

Pyruvate and ADP (10mM, 2mM, 5mM, ADP 5mM, respectively). OCR was monitored at

basal level and when the samples were treated with different drugs Oligomycin(5mM), FCCP

(1uM), and Antimycin (5mM).

Western Blot

Western blot were performed to detect target proteins using Ybx2 (Abcam), Gapdh (Abcam),

Ucp-1(Abcam), Pgc1α (Santa Cruz), Cidea (Santa Cruz), Pparϒ (Santa Cruz), P-AKT (Cell

signalling), AKT (Cell Signalling), Cpt1a (Proteintech), MCad (Santa Cruz) , β-actin (Sigma),

Tubulin (Cell signalling) antibodies.

Gene Ontology analysis and GSEA

Gene lists were analyzed for enrichment of Gene Ontology (GO) terms using DAVID

Functional Annotation Tools (31; 32). Gene set enrichment analysis (GSEA)(33) was

performed using default parameters with the pre-ranked gene sets.

RNA-decay analysis


Brown preadipocytes were cultured and differentiated for 5 days. We treated cells with

5ug/ml Actinomycin D (Sigma) and harvested RNA at different time points indicated in the

figures. We took the same proportion of RNA from each sample at different time point,

conducted reverse transcription with random primers and realtime PCR. CTs from each

sample were used to calculate the remaining percentage of mRNA at each point. We fit

these data into a first phase decay model to derive mRNAs’ half-life.

Yt=(Y0 - Plateau)*exp(-kdecay*t) + Plateau

Yt, the remaining percentage at a given time

Y0, the initial amount of RNA

t, time after transcription inhibition

kdecay, the rate constant

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was assessed using the

unpaired, 2-tailed Student t test. Statistical significance for samples with more than 2 groups

was determined by one-way ANOVA. The distribution difference between different

cumulative curves was determined by Kolmogorov-Smirnov Test. P values of < .05 were

considered to be significant.

ACCESSION NUMBERS

The accession number for the RNA-seq data reported in this paper is NCBI GEO:

GSE66686, GSE29899, GSE86590, GSE86338

RESULTS

Genome-wide identification of BAT-enriched RBPs

To identify RNA-binding proteins (RBPs) functionally important for BAT, we profiled the gene

expression of 413 RBPs annotated in the RBP database(18) in interscapular BAT, inguinal

WAT (iWAT) and epididymal WAT (eWAT), which led to identification of 26 BAT-enriched

RBPs. To further assess whether these RBPs are dynamically regulated during WAT

browning and brown adipogenesis, we examined their expression alternation during inguinal

WAT browning induced by a β3-agonist (CL-316, 243), and in primary brown preadipocytes

vs mature adipocytes. By intersecting these gene sets, we discovered 6 BAT-enriched RBPs

that were induced during browning and brown adipogenesis, including Pgc1β, Larp4,

Rbpms2, Grsf1, Akap1 and Ybx2 F (Fig. 1A-D), for further investigation.

Because Pgc1β is not a typical RBP, we excluded it from our subsequent experiments. For

the other 5 candidates, their tissue enrichment and dynamic regulation during WAT browning

were successfully validated by real-time PCR across 15 major mice organs (Fig. 1E) and in

inguinal WAT after housing animals for 7 days at 4oC (Fig. 1G). In BAT, only Ybx2 was

significantly induced upon cold treatment (Fig. 1F). To test whether these RBPs were


repressed upon BAT and beige fat inactivation, we housed mice at thermoneutrality (30oC)

for 7 days to induce “whitening” of BAT and iWAT. All 5 RBPs were down-regulated during

BAT and iWAT “whitening” (Fig 1H-I). Next, we examined their expression during an in vitro

differentiation time course of primary brown and white adipocyte culture. All 5 RBPs were

up-regulated during brown and white adipogenesis, with a higher expression level in brown

adipocytes (Fig. 1J). Finally, to test the human relevance of these observations, we

examined their expression across a differentiation time course of primary preadipocytes

isolated from human fetal interscapular BAT and subcutaneous WAT(27). The expression of

YBX2 and RBPMS2 increased throughout the human cell differentiation course with higher

levels in BAT adipocytes. AKAP1 exhibited a significant induction from Day 0 to Day 7 and

then decreased towards the end of differentiation, but its level was still higher in brown

adipocytes than white adipocytes (Fig. 1K).

To investigate the function of these 5 RBPs in brown adipocyte differentiation, we depleted

them by infecting brown preadipocytes with retroviral shRNAs and then induced cells to

differentiate for 5 days. Depletion of each of these RBPs resulted in distinct phenotypes.

Knocking down Ybx2 expression by ~90% (sh-3) severely blocked lipid accumulation (Fig.

2A) and reduced the expression of pan-adipogenic markers Fabp4 and PparΥ2, indicating a

block of pan-adipogenesis gene program, while inhibiting Ybx2 by ~70% (sh-1) affected BAT

markers but didn’t affect pan-marker expression and lipid accumulation (Fig.2B), suggesting

that the expression of BAT-selective genes is more sensitive to Ybx2 depletion. To

determine the role of Ybx2 in cellular respiration, we inhibited its expression by ~70% (sh-1)

in brown adipocytes, and used the Seahorse XFp Extracellular Flux Analyzer to measure the

oxygen consumption rate (OCR). A significant decrease of ORC for basal respiration and

proton leakage was observed (Fig. 2E).

While knockdown of Akap1 slightly reduced lipids accumulation (Fig. 2A), it didn’t affect pan-

adipogenic marker expression but the BAT-selective markers were down-regulated (Fig. 2C,

Fig S1A). Inhibiting Rbpms2 had a slightly influence on lipid accumulation (Fig. 2A) and pan-

adipogenic marker expression (Fig. 2D), but stronger effects on BAT-selective markers (Fig.

2D, Fig S1A). Consistently, OCR analysis showed a significant decrease of OCR attributed

to proton leak in the Rbpms2- and Akap1-depleted cells (Fig. S1B, C). In contrast, Inhibiting

Grsf1 and Larp4, didn’t affect lipid accumulation (not shown) or marker expression (Fig.

S2A-D).

Ybx2 is an essential regulator of brown adipocyte differentiation in vitro

Ybx2 harbors an ultra-conserved cold-shock RNA binding domain (CSD). Proteins bearing

CSDs, known as cold shock proteins, have been reported to regulate cellular adaptation

response, mainly at posttranscriptional levels, to cold stress in prokaryotes(34; 35). Because


BAT is a major organ for cold adaption in mammals, the presence of CSD in Ybx2 suggests

that Ybx2 may play a role in BAT thermogenesis. We validated the expression of Ybx2 at the

protein level by Western blot in different adipose depots (Fig. 4A) and during brown and

white adipogenesis (Fig. 2F). Consistent with its mRNA expression pattern, Ybx2 protein

level is higher in BAT and induced during differentiation. To determine its function in beige

adipocytes, we knocked it down in preadipocytes isolated from inguinal WAT, followed by

induction of differentiation, and observed a clear reduction of BAT markers (Fig. S2E-G). To

ensure the phenotypes of Ybx2 knockdown are not due to off-targeting effect, we further

targeted different regions in its mRNA using a different shRNA retroviral vector. Inhibiting

Ybx2 invariably impaired lipid accumulation and BAT marker expression in both BAT and

iWAT adipocyte cultures (Fig. S2H-L).

We next tested whether Ybx2 is sufficient to promote beige and brown adipogenesis by

overexpressing Ybx2 in primary white and brown preadipocytes with retroviral vector (Fig.

3A,D), followed by induction of differentiation. Ectopic expression of Ybx2 in white

adipocytes enhanced lipid accumulation assessed by bodipy staining (Fig. 3B) and

increased the expression of key BAT markers such as Ucp1 and Pgc1α (Fig. 3C).

Overexpression of Ybx2 in primary brown adipocyte culture also enhanced lipid

accumulation (Fig. 3E) and BAT marker expression (Fig. 3F) in the early phase of

differentiation (day 3), which was accompanied by a higher basal ORC and proton leakage

ORC (Fig3 G). Western blot showed elevated protein levels of Ucp1 and two fatty acid

oxidization regulators, Mcad and Cpt1a at day 3 of differentiation (Fig 3H). After 6 days of

differentiation, the expression of BAT markers in control cells caught up with that in the

Ybx2-overexpressing cells, probably because the abundance of endogenous Ybx2 at this

stage is sufficient to support full induction of the BAT-selective gene program. Taken

together, these observations indicate that Ybx2 can promote brown adipogenesis in white

adipocyte culture and accelerate brown adipogenesis in brown adipocyte culture.

Ybx2 is needed for full BAT development in vivo

To determine the function of Ybx2 in BAT in vivo, we imported Ybx2 knockout (KO) mice.

Knockout animals were infertile(36) but viable and born at expected Mendelian ratios. We

confirmed their lack of Ybx2 by Western blot (Fig. 4A). Knockout animals didn’t exhibit

significant alternation in their body weight (Fig. 4B), fat and lean mass (Fig. S3A, B). The

iWAT and eWAT of KO animals didn’t change significantly in size either (Fig. S3C, D),

whereas their iBATs were moderately but significantly smaller (Fig. 4B), coincident with

slightly smaller lipid droplets under microscope (Fig. 4C, D). To study the effect of Ybx2

knockout at the molecular level, we quantified the expression of pan-adipogenic and BAT-

selective marker genes by real-time PCR, and observed no change in pan-adipogenic

markers (Fig. S3E) but a detectable decrease in Ucp1, Prdm16 and Dio2 (Fig. S3F). RNA-


seq was performed to examine the global effects of Ybx2 KO on gene expression, but very

few genes showed significant difference (Supplemental File 2), indicating that Ybx2 is

dispensable for BAT to maintain its gene-expression program at room temperature. In iWAT,

we didn’t observe significant change of BAT-selective markers as well as a WAT-marker,

HoxC10 (Fig. S3G). Glucose tolerance test (GTT) revealed a glucose intolerance (Fig. S3H);

Insulin tolerance test (ITT) detected a trend of insulin intolerance but the difference was not

statistically significant (Fig. S3H). Nevertheless, to what extent the impaired glucose

tolerance can be accounted by a smaller BAT or by systemic effects from other organs

needs to be investigated in the future.

Since whole-body Ybx2 deficiency may have indirect effects on BAT phenotypes, to confirm

whether Ybx2 knockout may exert cell-autonomous effect, we isolated brown pre-adipocytes

from KO and WT mice for differentiation. Real-time PCR revealed decreased expression of

pan-adipogenic markers and a more significant reduction of BAT-selective markers in the

KO cells (Fig. S4A-C), consistent with the shRNA knockdown phenotypes. RNA-seq was

then performed to profile the genome-wide effect of Ybx2 KO, and gene set enrichment

analysis (GSEA) revealed that the pathways of adipogenesis, fatty acid oxidation, oxidative

phosphorylation and cellular respiration were significantly down-regulated (Fig. S4D). Thus,

Ybx2 should have cell autonomous effects on brown adipocyte differentiation in vitro but

such an effect was much ameliorated in vivo.

Ybx2 is required for cold-induced BAT activation

To determine the role of Ybx2 in BAT activation, we exposed WT and KO animals to 4oC for

6 hours. The WT BAT mass upon cold activation became smaller than that at room

temperature, but the KO BAT mass didn’t decrease after cold exposure (Fig. 4E).

Consistently, Hematoxylin and Eosin staining revealed that lipids in WT BAT but not the KO

BAT were largely depleted (Fig.4F-G), indicating that the KO BAT failed to combust lipids

upon cold activation. To directly assess the effects of Ybx2 KO’s function, we measured the

OCRs for cold-activated WT and KO BAT with Oroboros respirometry. We observed a

decreased OCR in KO BAT before but not after Fccp treatment, which suggested that loss-

of-Ybx2 didn’t change the maximal OCR capacity but reduced the cold-provoked

mitochondria activity (Fig. S5A). Consistently the core body temperature of KO mice dropped

faster than that of WT animals at cold temperature (Fig. 4H). Although the BAT defect is a

certain culprit of the cold intolerance, we can not preclude the possibility that the effect of

Ybx2 KO on other organs can also contribute to this phenotype.

We examined the lipolysis rates in the WT and KO BAT and didn’t observe any significant

change (Fig S5B), indicating that the larger BAT mass in the KO BAT is unlikely due to any

change in lipolysis. In addition, to test whether loss-of-Ybx2 affects insulin signaling in BAT,

we performed Western blot to detect p-AKT in BAT and found that cold challenge could


enhance the insulin sensitivity in WT but not in KO BAT (Figure S5C). To test whether Ybx2

KO may affect BAT-selective gene expression in beige adipocytes, we performed real-time

PCR for iWATs and found a significant down-regulation of Ucp1 but not other detected

markers (Figure S5D).

To examine the effect of Ybx2 on BAT activation at the molecular level, we conducted RNA-

seq of BAT isolated from WT and KO mice at both room temperature and after cold

activation. One of the most striking observations was that the cold-induced thermogenic

program in BAT was severely hindered in KO animals. Ucp1, Dio2, Pgc1α, and Elvol3 were

among the most significantly depleted genes in KO upon cold exposure (Fig. 5A, S6A),

which we validated by real-time PCR and Western blot (Fig. 5C, D). Consistent with the

individual markers, pathways analysis revealed that one of the most enriched pathways

associated with the down-regulated genes is oxidation reduction (Fig. 5B).

To integrate the gene expression profiles at room temperature and after cold exposure, we

calculated the fold change of each gene after cold exposure in both WT and KO BAT (Fig.

S6B), and looked for enriched pathways among the most differentially regulated genes. The

mitochondrion and fatty acid metabolic process pathways were among the top down-

regulated pathways (Fig. S6C). Importantly, the BAT mass and gene expression changes in

KO animals were not gender-dependent and were also observed among female animals (Fig.

S7A-D). Thus, although BAT can still form in the absence of Ybx2, its thermogenic response

to cold temperature is impaired.

As a part of BAT adaptation to cold exposure, glucose uptake, lipogenesis and combustion

of long chain fatty acids (LCFAs) are increased in coordination with stimulation of β-oxidation

and thermogenesis(37; 38). In Ybx2 KO BAT, besides thermogenic genes, those involved in

glucose uptake (Glut4), lipogenesis (Scd1, Fasn, Dgat1, Dgat2, Acaca) and long chain fatty

acid generation (Elvol3, Elvol6) were also reduced (Fig. 5E, S7E), which was further

supported by pathway analysis (Fig. 5B, S6C). Thus, Ybx2 is a regulator orchestrating

glucose metabolism, lipid metabolism and thermogenesis during BAT activation.

Ybx2 stabilizes mRNA targets encoding proteins enriched for mitochondrial functions

To identify the mRNA targets of Ybx2, we performed RNA-immunoprecipitation followed by

RNA-seq (RIP-Seq) using an antibody against Ybx2 in both brown and white adipocyte

culture (Fig. S8A). First, we confirmed the successful Ybx2 precipitation by Western blot (Fig.

6A). We then selected candidates with at least 8-fold enrichment in the Ybx2 IP sample

compared with IgG control, which revealed 800 and 1822 potential mRNA targets in BAT

and WAT, respectively. Targets in BAT and WAT significantly overlapped, leading to

identification of 414 common targets (Fig. 6B). As expected, Ybx2 can target many mRNAs

encoding proteins involved in post-transcriptional RNA processing, a general feature of


RBPs(39-42). Functional terms enriched among Ybx2 targets include ribosome,

ribonucleoprotein complex and translation (Fig. 6C). Interestingly, these targets were also

enriched for mitochondria term (Fig. 6C). To further confirm this observation, we calculated

the relative abundance of each mRNA in Ybx2 vs IgG sample and plotted the cumulative

distributions for mitochondrion-related genes (206 genes) as well as for all genes detectable

in the RIP-seq assay (4095 genes). The cumulative curve of mitochondrion significantly

shifts towards the right (Fig. 6D), confirming that Ybx2’s targets are enriched for

mitochondrial functions.

Next, we asked if Ybx2 exerts a functional impact on its mRNA targets. Utilizing RNA-seq

data, we calculated the fold change of each mRNA between KO vs. WT BAT, and plotted the

cumulative distributions for Ybx2 target and non-target mRNAs. At room temperature,

targets and non-targets distributions didn’t show significant difference (Fig. 6E), but upon

cold activation, Ybx2’s targets were markedly repressed in KO BAT (Fig. 6F). These data

support a role of Ybx2 in stabilizing its target mRNAs, which was suggested by earlier work

in ooctyes(43).

Ybx2 targets and stabilizes Pgc1α mRNA

Among the top Ybx2 targets was the Pgc1α mRNA that is significantly decreased in KO BAT.

We used Pgc1α as an example to illustrate how Ybx2 recognizes and affects its targets. To

confirm binding between Pgc1α mRNA and Ybx2 in vivo, we performed the RIP-PCR in

tissue lysate from KO and WT BAT to detect Pgc1α mRNA precipitated by Ybx2. A clear

reduction of Pgc1α signal was detected in RIP from KO BAT, while such a reduction was not

observed for Fabp4 mRNA, which bears a short 3’UTR (180bp) and is used as a control (Fig.

7A). To dissect Ybx2-binding sites within Pgc1α mRNA, we performed RNA-pulldown assay

using 4 in vitro transcribed sequential RNA fragments from Pgc1α 3’UTR and found that a

1101nt RNA fragment (fragment 3) can readily retrieve Ybx2 protein (Fig. 7B). We then

generated 8 small RNA fragments (200-300bp) from this segment for a second round of

pulldown assays, and found that fragments 3.1, 3.3, 3.5 and 3.7 can retrieve Ybx2 (Fig. 7C).

Intersecting segments 3.1 vs. 3.5, and 3.3 vs. 3.7 locate two Ybx2-binding sites-harboring

regions. Further truncation of these two fragments abolished their interactions with Ybx2

(data not shown), suggesting that a secondary or tertiary nucleotide structure may be

necessary for Ybx2 binding. To test whether this identified RNA fragment can define the

Ybx2 binding site in human, we blasted the human Pgc1α 3’UTR and mouse Pgc1α 3’UTR

and identified a ~1kb segment with >90% homologous to the fragment 3 in Figure 7B (Fig

7D). We cloned this fragment for pulldown assay. As expected, this fragment can retrieve

Ybx2 in BAT lysate (Fig 7D), indicating that the Ybx2-Pgc1α interaction is conserved.

To test whether the interactions between Ybx2 and Pgc1α is enhanced at cold exposure, we

performed RIP-PCR in BAT before and after cold exposure, and found that Ybx2 could


retrieve more Pgc1α mRNAs upon cold exposure (Fig 7E). To further study whether this

apparent increase is due to an enhanced binding affinity or an elevated Pgc1α mRNA

abundance upon cold exposure, we inhibited transcription with Actinomycin D in cultured

brown adipocytes and then performed RIP-PCR to detect the Ybx2-Pgc1α mRNA interaction

in the presence or absence of norepinephrine treatment. Interestingly, in the Actinomycin D

treatment cells, the Pgc1α mRNA retrieved by Ybx2-antibody was similar before and after

norepinephrine treatment (Figure S8B). Therefore, BAT activation likely increases the Ybx2-

retrived Pgc1α mRNA by stimulating Pgc1α mRNA expression but not by changing their

binding affinity.

To examine the influence of Ybx2 knockout on Pgc1α mRNA stability, we used Actinomycin

D to stop mRNA transcription in WT and KO brown adipocyte culture and measured the

decay rates for Pgc1α and Fabp4 mRNA. The half-life of Pgc1α mRNA decreased from 2.39

to 1.29 hours in the absence of Ybx2 (Fig. 7F), supporting a role of Ybx2 in stabilizing Pgc1α

mRNA. To investigate whether the above identified Ybx2-binding sites in Pgc1α 3’UTR can

mediate the mRNA-stabilizing effect from Ybx2, we constructed two reporter plasmids: one

containing a ~2kb Pgc1α 3’UTR after the renilla luciferase (WT) and another containing a

truncated 3’UTR without the Ybx2-binding fragment (Mutant). We measured the decay rates

of the renilla luciferase mRNA in 293 cells in the presence and absence of a Ybx2-

expressing vector. In the absence of Ybx2, both reporter constructs manifested similar decay

rates (Fig S8C); in the presence of Ybx2, the mRNA decay rate of the WT reporter is ~2-fold

slower than that of the mutant reporter (~8.4 hours vs. ~4.1 hours), indicating our identified

Ybx2-binding sites (Fig 7B,C) in the Pgc1α 3’UTR is required for Ybx2’s mRNA stabilization

function.

We further examined the functional interactions between Ybx2 and Pgc1α by overexpressing

a full length ORF Pgc1α in the Ybx2-inhibited brown adipocytes. As described above (Fig

2B), knockdown of Ybx2 reduced BAT marker expression, but Pgc1α overexpression could

significantly rescue the phenotype (Fig 7G). Therefore, although stabilizing Pgc1α alone is

unlikely to account for all the phenotypes of Ybx2 KO, Ybx2 may be a key target of Ybx2 and

Ybx2’s function at some extent relies on Pgc1α expression.

DISCUSSION

Early studies have suggested a role of Ybx2 in global mRNA stabilization. Schultz group

knocked down Ybx2 in oocytes by expressing a transgenic Ybx2 hairpin dsRNA. They

observed 60% reduction of Ybx2 protein and 75-80% reduction of poly-(A) mRNAs(44). In

another study, they generated a knockout strain and observed severe defects in

spermatogenesis and oocyte development(36), accompanied by a ~25% decrease of

mRNAs in the mutant oocytes(43). Exogenous mRNAs injected into mutant oocytes were


lower than that in wild-type cells, consistent with a decreased mRNA stability in the absence

of Ybx2(43). This is consistent with our conclusion which demonstrated a role of Ybx2 in

enhancing mRNA stability in a more systemic manner (Fig 6F).

As a RBP, Ybx2 may affect multiple RNA processing steps including but not limited to RNA

stability. Given its cytosol localization (Fig S9C), it is not surprising if Ybx2 can influence

translational control for certain mRNAs. This may explain why the changes in mRNA and

protein levels for some genes, to some extent, may display discordance. The potential

influence of Ybx2 on translation will be further studied in the future.

Skeletal muscle is known to contribute to non-shivering thermogenesis (NST) mainly through

Sarcolipin-mediated ATP-hydrolysis by SERCA(45; 46). To examine whether Ybx2 KO can

alter this pathway, we examined the NST genes in muscles. Despite an increase of

Sarcolipin expression observed in KO muscle, Ybx2 KO did not affect Serca1-3 expression

(Fig S9A) and OCRs(Fig S9B) directly measured by Oroboros respirometry. Therefore,

although it is unclear whether the increase of Sarcolipin expression in muscle is due to a

tissue autonomous effect or a cross-organ response to the compromised KO BAT, the NTS

function of muscle was not altered.

In sum, we profiled the expression of >400 RBPs across different fat depots, during

adipogenesis and WAT browning, and identified Ybx2, a CSD-containing protein that

orchestrates BAT activation. CSD-containing proteins are among the most phylogenetically

conserved families and are known for their role in cold adaptation in prokaryotes (34; 35).

Because BAT activation is a part of cold adaptation in mammals, we speculated that CSD

proteins, exemplified by Ybx2, may be evolutionarily conserved to mediate cold adaptation at

the whole organismal level via roles in BAT activation.


FIGURE LEGENDS

Fig. 1. Genome-wide identification of BAT-enriched RBPs. (A-C) Gene expression of

RBPs by RNA-seq in (A) BAT, iWAT and eWAT, (B) during iWAT browning, and (C) primary

brown preadipocyte and mature adipocytes. Heatmaps showed the row mean-centred

abundance. (D) Selection of gene expression from profiling studies A-C, plotted in Venn

diagrams. (E) Real-time PCR validation of gene expression for 5 RBPs across 15 mouse

organs. Heatmap shows the row mean-centered expression. (F) Gene expression of RBPs

by real-time PCR in BAT and (G) iWAT after housing mice (8-weeks old) at 4oC for 7 days.

n=6 (H, I) Gene expression of RBPs by real-time PCR in (H) BAT and (I) iWAT after housing

mice (8 weeks old) at 30oC for 7 days. Mouse housed at RT (Room temperature) was used

as control group n=5 per group. (J) Gene expression of RBPs during the differentiation of

mouse primary brown and white adipocyte cultures. n=4. (K) Gene expression of RBPs by

real-time PCR during in vitro differentiation of stromal vascular fraction (SVF) cells isolated

from human fetal BAT and subcutaneous WAT. n=4. Error bars are mean ± SEM, *p<0.05,

Student’s T test.

Fig. 2. Ybx2 is an essential regulator of brown adipocyte differentiation in vitro.

(A) Primary brown pre-adipocytes were infected by retroviral shRNAs targeting RBPs, Ybx2,

and Akap1, followed by induction of differentiation for 5 days. Oil-Red O staining was used to

assess lipid accumulation. (B-D) Real-time PCR to measure the knockdown efficiency (left),

pan-adipogenic marker expression (right), and BAT-selective marker expression (bottom) in

cultured primary brown adipocytes (Day 5) infected by retroviral shRNAs targeting Ybx2 (B),

Akap1(C) and Rbpms2 (D). n=3. Error bars are mean ± SEM, *p<0.05, one-way ANOVA. (E)

Representative metabolic flux curves from cultured brown adipocytes (Day 5) infected by

retroviral shRNA targeting Ybx2. Cells were sequentially treated with Oligomycin, FCCP,

Retenone. Oxygen consumption rates (OCR) are normalized by protein concentration. n=5.

Error bars are mean ± SEM, *p<0.05, Student’s T test. (F) Western blot to examine the

protein levels of Ybx2 during primary brown and white adipocyte differentiation in culture.

Fig. 3. Ybx2 can promote BAT-selective gene expression in white and brown

adipocyte cultures. (A) Western blot to confirm the overexpression of Ybx2 in primary white

adipocyte culture. (B) Representative picture of Bodipy staining for lipids in primary white

adipocytes infected by Ybx2-expressing or empty vector. (C) Real-time PCR to examine

marker gene expression during the time course of white adipocyte cultures expressing Ybx2

or vector. n=4. (D-F) Same as in (A-C), but in primary brown adipocyte culture. n=4. Error


bars are mean ± SEM, *p<0.05. (G) Representative metabolic flux curves from cultured

brown adipocytes (Day 3) infected by retroviral overexpressing Ybx2. Cells were sequentially

treated with Oligomycin, FCCP, Retenone. Oxygen consumption rates (OCR) are

normalized by protein concentration. n=5. (H) Western blot to detect the protein levels of

Ucp1, and two FAO components Cpt1a and Mcad at day 3.

Fig. 4. Ybx2 is needed for cold-induced BAT activation. (A) Western blot to detect Ybx2

expression in eWAT, BAT and iWAT from WT and KO mice. (B) Body weight and BAT organ

weight of WT and KO male mice at 8-9 weeks old. WT n=6; KO n=7. (C) Representative

picture of H&E staining under the microscope of WT and KO BAT. (D) Distribution of the

diameters of lipid droplets from (C) measured by Image J software. (E) Body weight and

BAT weight of 8-9 week old WT and KO animals after 6 hours at 4oC exposure. n=5. (F)

Representative picture and H&E staining under microscope of BAT from WT and KO mice

after cold exposure. (G) Distribution of the diameters of lipid droplets from (F). (H) Body

temperature was measured by rectal probe at the indicated times at 4oC. n=5. Error bars are

mean ± SEM, *p<0.05.

Fig. 5. The effect of Ybx2 knockout on cold-induced gene expression in BAT.

(A) Heatmap of the gene expression in WT and KO BAT after 6 hours cold exposure.

Heatmap showed the row mean-centred abundance. (B) 5 top non-redundant gene ontology

(GO) terms enriched among mRNAs that showed significantly low (top) or high (bottom)

expression (p<0.05, CUffdiff) in KO vs. WT BAT. (C) Real-time PCR to confirm gene

expression of BAT-selective genes in WT and KO BAT. n=5. (D) Western blot to confirm

gene expression of BAT markers in WT and KO BAT. (E) Real-time PCR to confirm the

expression of genes involved in lipogenesis and glucose uptake. n=5. Error bars are mean ±

SEM, *p<0.05.

Fig. 6. Ybx2 stabilizes mRNA targets encoding proteins enriched for mitochondria

functions.

(A) Western blot to confirm immunoprecipitation of Ybx2 protein by Ybx2 antibody. 10% IP

cell lysate was used as the input. (B) Targets of Ybx2 were selected based on their

enrichment in the Ybx2 IP vs. IgG control. Venn diagram showed the overlapping of

candidates from brown and white adipocytes. (C) Bubble chart to show the GO terms

enriched in the common targets. X-axis indicates P values, Y-axis indicates the enrichment

score. The bubble size indicated the number of targets in that GO category. (D) Relative


abundance of each mRNA was calculated in Anti-Ybx2 vs IgG RIP-seq. The cumulative

fraction of mRNAs involved in mitochondrion and all other detectable genes were plotted.

Kolmogorov–Smirnov test was performed to determine the distribution difference. (E)

Relative expression of each gene in KO vs. WT BAT at room temperature based on RNA-

seq data. The cumulative fraction curves were plotted for 414 common target mRNAs and

other genes detectable in the RIP-seq assays. (F) The cumulative fraction curves were

plotted for common target mRNAs and other genes after cold exposure. Kolmogorov–

Smirnov test was performed to determine the statistical significance of the difference in the

distributions.

Fig. 7. Ybx2 binds and stabilizes Pgc1α mRNA. (A) RIP assay with anti-Ybx2 in brown

adipose tissue lysate from WT and KO animals to examine the amount of Pgc1α mRNA in

the IP samples. Fabp4 was used as a control. 5% tissue lysate in the IP reaction was used

as the input. n=3. (B,C) RNA pulldown assay was conducted to determine which RNA

segments from Pgc1α 3’UTR can bind Ybx2. Segments in 3’UTR as shown in the diagram

were cloned for in vitro transcription to generate RNA fragments which were used for RNA-

pulldown assay in BAT lysate, followed by Western blot to determine presence of Ybx2 in

each pulldown reaction. An AU-enriched ~100nt fragment from androgen receptor (AR) was

used as a negative control. (D) RNA pulldown assay was conducted using a ~1kb fragment

from human Pgc1α 3’UTR that is homologous to the fragment 3 in (C). (E) RIP-PCR was

conducted to determine Pgc1α mRNA retrieved by anti-Ybx2 in BAT from RT and Cold-

exposed animals (n=3). (F) Primary brown preadipocytes were isolated from WT and KO

BAT for culture and then induced to differentiate for 5 days (left). Actinomycin D was added

to stop transcription, and RNAs were harvested at the indicated time points (X-axis) after

transcription inhibition. Real-time PCR was carried to determine remaining RNA level

compared to the starting time point. The trajectory of Pgc1α mRNA was fit into a first order

decay curve to derive the RNA half-life (WT T1/2=2.39 hours; KO T1/2=1.29 hours). Fabp4

mRNA was used as a control. n=6. (G) We used retroviral constructs to knock down Ybx2

and overexpress Pgc1α in primary brown preadipocytes, followed by induction of

differentiation. BAT-selective markers were examined by reat-time PCR at day 6 (n=4, Error

bars are mean ± SEM, *p<0.05.)


ACKNOWLEDGEMENTS

Thanks to Dr.Paula Stein, University of Pennsylvania, for the KO mice as a generous gift.

Thanks for Dr. Manvendra Singh, Duke-NUS Medical School, for the coordination of mice

transportation. This work was supported by Singapore NRF fellowship (NRF-2011NRF-

NRFF 001-025) to L.S. This research is also supported by the Singapore National Research

Foundation under its CBRG grant (NMRC/CBRG/0070/2014 and NMRC/CBRG/0101/2016)

and administrated by the Singapore Ministry of Health's National Medical Research Council.

Dr. Lei SUN is the guarantor of this work and, as such, had full access to all the data in the

study and takes responsibility for the integrity of the data and the accuracy of the data

analysis

AUTHOR CONTRIBUTIONS

X.D., X.S, K.A.M.M., L.Y.C, C.S.Y., and A-D.J.R., performed experiments. S.L. and X.D.

designed experiments and wrote the manuscript. M.L. and C.P. discussed the experiment

design and critically reviewed the manuscript.

CONFLICTS of INTEREST

The authors declare no conflicts of interest.


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Ybx2 Days

β-actin

TubulinB tubulin

During cell differentiation

BTubulin

Cold induced multiple western blot

BAT WAT

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

VectorSh-Ctl

Control

Rbpms2

Sh-1 Sh-2 Sh-3

Sh-1 Sh-2 Sh-3

Ybx2

Akap1

Sh-1 Sh-2 Sh-3


0 3 60

10

20

30

40

A B

C

D E

Vector Ybx2

β-actin

Ybx2

100µm 100µm

Vector Ybx2

100µm 100µm Vector Ybx2

100µm 100µm Vector

Ybx2

Ybx2

β-actin

0 2 4 6020406080100

20003000

Cidea

0 3 60

10

20100200

Ucp1

0 3 60

5

10

15

20Pgc1αPparϒ2

Rel

ativ

e ex

pres

sion

* **

*VectorYbx2

F

Figure 3

0 3 60

50

100

150

0 3 601020304050

0 3 60

2

4

6

0 3 60

50

100

150

200

0 3 60

50

100

150

200

0 3 60

10

20

30

40

0 3 6050100150200250

Ucp1

Prdm16

Pparϒ2

Pparα

Pgc1α

Cox4

Cidea

VectorYbx2

**

Rela

tive

expr

essi

onRe

lativ

e ex

pres

sion

* * **

* * ** *

0 20 40 60 80 1000

10

20

30

40

50

OC

R (p

Mol

/(min

*µg)

)

Ybx2

ControlOligomycin

FCCP Rotenone

Vector Ybx2

FAO and UCP1 Western blot in overexpression cells

MAcd

B actin

UCP-1

CPT1A


MAcd

B actin

UCP-1

CPT1AFAO and UCP1 Western blot in overexpression cells

MAcd

B actin

UCP-1

CPT1A


MAcd

B actin

UCP-1

CPT1A

Cpt1aUcp1Mcad

β-actin

G H

0246810

0

2

4

6*

012345 *

BasalProton leak Maxi

ATP turnover

OC

R (p

Mol

/(min

*µg)

)

0

10

20

30

40


WT KO0.0

0.2

0.4

0.6

0.8

1.0Fa

t mas

s (%

BW)

WT KO25

30

35

40

45

50

Bod

y w

eigh

t (g)

WT KO0.0

0.2

0.4

0.6

0.8

1.0

Fat m

ass(

%B

W)

10 20 30 40 50 60 70 80 90100 10 20 30 40 50 60 70 80 9010

00.00.10.20.30.40.5 WT KO

Rea

tive

fract

ion

WT KO

Body weight*

BAT

Figure 4

A

B

WT KO

Ybx2β-actin

WT KO

Body weight iBAT*

0 2 4 630

32

34

36

38

40 WTKO

Cold exposure (h)

Body

tem

pera

ture

(o C)

* *

D

C

H

E

F

G

10 20 30 40 50 60 70 80 90100 10 20 30 40 50 60 70 80 9010

00.0

0.1

0.2

0.3

0.4

Rea

tive

fract

ion

WT KO

Diameter (arbitrary units)

Diameter (arbitrary units)

WT KO20

30

40

50

Fat m

ass

(%BW

)


Ucp1

Pgc1α

Pparα

Prdm16

Elovl3

Dio2Cide

a0.0

0.5

1.0

1.5

* * **

Rel

ativ

e ex

pres

sion

WTKO

Mogat1

Acaca

Srebf1

Dgat1

Fasn

Elovl6

Dgat2

scd1

Glut4

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e ex

pres

sion

**

*

** *

* *

E

A B

C

Scd1Ucp1Fasn

Dgat2

Ppargc1a

Acaca

Slc2a4Dio2

Dgat1

Elovl3

Ybx2

Tob1

Acss2

Scd1Ucp1Fasn

Dgat2Pgc1α

AcacaSlc2a4

Dio2Dgat1

Elovl6Elovl3

Cold 6 hours

FPKM

cha

nge

50

-50

0

0 5 10 15 20 25

0 5 10 20 25 30

Lipid biosynthetic process

Glucose metabolic process

Oxidation reduction

Triglyceride metabolic process

Phospholipid biosynthetic process

-log2(P-value)

Blood vessel development

Cell adhesion

Cell motion

Cytoskeleton

Actin binding

-log2(P-value)

Dow

n-re

gula

ted

GO

Up-

regu

late

d G

O

D

β-actin

Pparγ

Ucp1

Pgc1α

Cidea

WT KO

B tubulin

During cell differentiation

BTubulin

Cold induced multiple western blot

BAT WAT

Tubulin

Figure 5


Figure 6

Ybx2

IP Input

IgG

Anti-Y

bx2

Input

IgG

Anti-Y

bx2

Gapdh

386 414 1408

WAT BAT

0

2

4

6

8

0 5 10 15 20 -log10(P-value)

Enr

ichm

ent translation

ribonucleoprotein complex

endomembrane system

25

40 mitochondria

A B

C D

E

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80

100

log2(KO/Wt)

Cum

mul

ative

per

cent

age

TargetsOther genes

Room temperature

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80

100

Cum

mul

ative

per

cent

age

log2(KO/Wt)

TargetsOther genes

cold

P=2.5e-11

-4 -2 0 2 4 60

20

40

60

80

100 BackgroupMitochondrion

-4 -2 0 2 4 60

20

40

60

80

100 BackgroupMitochondrion

log2(anti-Ybx2/IgG)

Cum

ulat

ive

Per

cent

age

P=0.0115

F

BAT WAT


Pgc1a

Fabp4

0

5

10

15WTKO

(IP/Input)%

Dio2

Gapdh

Pgc1a

0

5

10

15

20WTKO

(IP/Input)%

*

A

F

D

B

Figure 7

CDS 3’UTR1 2 3 4

3.8 kbNM_008904.2

Ybx2

Gapdh

3.1

3.53.6

3.23.3

3.73.8

3.4

3Ybx2 binding site1 Ybx2 binding site2

317nt 335ntC

0 2 4 6 8 100

20

40

60

80

100

KO

WT

Rem

aini

ng P

erce

ntag

e

Hours

WT T1/2 = 2.39 hr

KO T1/2 = 1.29 hr

0 2 4 6 8 100

20

40

60

80

100

KO

WT

Rem

aini

ng P

erce

ntag

e

Hours

WT T1/2 = 2.39 hr

KO T1/2 = 1.29 hr

Ybx2

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion *

*

Ucp1

Pparα

Prdm16

Cidea

Dio2Cox

70.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

**

**

**

**

**

**

Sh-Ctlsh-Ybx2shYbx2+Pgc1α

G

E

HUMAN PULL DOWN

YBX2

Gapdh

HUMAN PULL DOWN

YBX2

Gapdh

Ybx2

Gapdh

0 2 4 6 8 100

50

100

Rem

aini

ng P

erce

ntag

e

Hours

Fabp4

0

5

1050

75

100

Input IgG Anti-Ybx2

RT

Cold

*

CDS

CDS

mouse

humanNM_013261.4

Rel

ativ

e Ex

pres

sion

*

*


RNA-seq data analysis RNA-seq was performed in the Illumina HiSeq2000 platform. Sequencing reads were first quality checked with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), subsequently aligned to mm10 using Tophat (version tophat-2.0.9). Aligned reads were then qualified using Cuffdiff (Version 2.1.1), which also performs statistical tests between tested conditions. Genes with low expression, defined by FPKM<0.1 in both tested conditions, were removed. Complete list of RBP was downloaded from http://rbpdb.ccbr.utoronto.ca/help.php. A RBP was considered significantly differentially expressed if (i) q-value<0.05 and (ii) more than 1.5 fold change between tested conditions. RIP-seq analysis RNA-seq analysis was performed as described above to obtain FPKM values for each gene. Genes with FPKM<1 in the RNA-seq from brown adipocytes were considered as undetectable in cells and were excluded. Genes with FPKM <5 in both IgG and anti-Ybx2 RIP-seq were regarded as unbound by Ybx2 and IgG and therefore also excluded. 5227 and 5883 genes passed these filters in WAT and BAT RIP-seq data for downstream analysis. The gene expression ratios between anti-Ybx2 and IgG were calculated as an indicator for the enrichment of each transcript by Ybx2 RIP. Because low FPKM values will introduce large noises to the ratios between anti-Ybx2 and IgG, RIP-seq data were adjusted by adding 0.5 FPKM on all genes before the ratios between anti-Ybx2 and IgG were derived. SUPPLEMETNAL FIGURE LENGENDS: Figure S1 (A) Western blots to detect the Ucp1 change in cultured primary brown adipocytes (day 5) where AKAP1 and RBPMS2 were knocked down by retroviral shRNA. (B, C) Metabolic flux curves from cultured brown adipocytes (Day 5) where AKAP1 and RBPMS2 were knocked down by retroviral shRNA. Oxygen consumption rates (OCR) are normalized by protein concentration. n=8. *p<0.05, one-way ANOVA. Figure S2. (A-D) Real-time PCR to examine the knockdown efficiency and marker expression in brown adipocytes expressing shRNAs targeting (A, B) Grsf1 and (C,D) Larp4. n=3. Error bars are mean ± SEM, *p<0.05, student’s t test. (E-G) Real-time PCR to measure the (E) knockdown efficiency, (F) pan-adipogenic markers and (G) BAT-selective markers in cultured primary white adipocytes (Day 5) that were infected by retroviral shRNAs targeting Ybx2. n=3. Error bars are mean ± SEM, *p<0.05, student’s t test. (H-K) Primary brown preadipocytes were infected by retroviral shRNAs (pMKO vector) targeting different regions of Ybx2 mRNA, followed by induction of differentiation for 5 days. (H) Oil-Red O staining (top) and TAG quantification (bottom) were used to assess lipid accumulation. Real-time PCR was performed to examine the (I) knockdown efficiency, (J) pan-adipogenic markers and (K) BAT-selective markers. (L) Similar to I-K, but in primary white adipocyte culture. n=4. Error bars are mean ± SEM, *p<0.05. one-way ANOVA Figure S3. (A) Fat and (B) lean mass of male mice measured by EchoMRI. (C, D) Organ weight of iWAT and eWAT in WT and KO male mice at 9 weeks old. n≥6. Error bars are mean ± SEM, *p<0.05, student’s t test.


(E) Real-time PCR of pan-abiogenic markers and (F) BAT-selective markers in WT and KO BAT. n≥7. (G) Real-time PCR of marker expression in iWAT. n≥6. Error bars are mean ± SEM, *p<0.05, student’s t test. (H) Blood glucose levels during glucose tolerance test (n=5) and insulin tolerance test (n=10), 16 weeks old male animals. Error bars are mean ± SEM, *p<0.05, student’s t test. Figure S4. (A-C) primary brown preadipocytes were isolated from WT BAT and KO BAT for in vitro culture and differentiation for 5 days. Real-time PCR was used to confirm the (A) knockdown efficiency (B) pan-adipogenic markers (C) BAT-selective markers. n=3, Error bars are mean ± SEM, *p<0.05. Student’s t test. (D) Genes were pre-ranked by their relative expression between KO and WT, followed by GSEA analysis. Figure S5. (A) The OCRs of WT and KO BAT were measured with Oroboros respirometry after housing 8-9 weeks old animals at 4oC for 6 hours. n=6, Error bars are mean ± SEM, *p<0.05. Student’s t test (B) Lipolysis assay to assess the lipolysis rate of WT and KO BAT isolated from animals exposed to acute cold temperature. n=6. (C) 8-9 weeks old Ybx2 KO and WT male mice were fasted for 6 hours at RT or 4OC. Insulin (1 U per kg body weight) were injected into these animals. Mice were then sacrificed after 5 mins injection. Brown adipose tissue were collected. Western Blot was performed to detect protein levels of P-AKT and AKT in BAT. (D) Real-time PCR to examine BAT-selective markers in iWAT after acute cold exposure. n≥9, *p<0.05, Student’s t-test. Figure S6. (A) FPKM of thermogenic markers in WT and KO BAT at room temperature and after cold treatment. (B) The fold changes (FC) of gene expression upon cold exposure were calculated for WT and KO BAT. The genes with more than 2 fold difference in FC were plotted in heatmap, and the color code represents the column mean-centered FC. (C) Pathway analysis was performed using DAVID Tools. Figure S7 (A) Body weight, (B) BAT mass (C) iWAT mass of female mice at 8-9 weeks after 6-hours cold challenge. n=6 (D) Real-time PCR to examine BAT-selective and (E) lipogenesis markers in BAT from female mice upon cold treatment (4oC,6 hours). 9 weeks old, n=6. Error bars are mean ± SEM, *p<0.05, student’s t test. Figure S8 (A) Diagram of the RIP-seq experiments. (B) RIP-PCR analysis to detect the Ybx2-Pgc1a mRNA interaction in differentiated brown adipocytes which was treated with NE for 6 hours in the presence of Actinomycin D. (C) 293 cells were transfected with a reporter plasmid (psi-Check2) harboring a ~2kb WT Pgc1a 3’UTR or a mutant plasmid without the Ybx2 binding sites in the presence or absence of YBX2. Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated time points (X-axis) after transcription inhibition. Real-time PCR was carried to determine remaining RNA level compared to the starting time point. The trajectory of Pgc1a mRNA was fit into a first order decay curve to derive the RNA half-life. n=4. Figure S9 (A) WT and KO animals (8-9 weeks old) were housed at 4oC for 6 hours. Skeletal muscle tissues (Gastrocnemius) were harvested for real-time PCR analysis. (B) The OCRs of muscle lysates were measured with Oroboros respirometry. n=6, Error bars


are mean ± SEM, *p<0.05, Student’s t-test. (C) Western blot to examine the cellular distribution of Ybx2 in BAT nuclear vs. cytosolic lysate.


SUPPLEMENTAL FILES Suppl file1_oligo sequences Suppl file2_BAT_WT and KO_RNA-seq https://www.dropbox.com/s/olt27q7d2o2i1jo/Suppl%20file2_BAT_WT%20and%20KO_RNA-seq.xlsx?dl=0 Suppl file3_AdipocyteD5_WT and KO_RNA-seq https://www.dropbox.com/s/1u4y0p0kk6jjt4c/Suppl%20file3_AdipocyteD5_WT%20and%20KO_RNA-seq.xlsx?dl=0 Suppl file4_RIP-seq and targets https://www.dropbox.com/s/3s21ch019fi4zqm/Suppl%20file4_RIP-seq%20and%20targets.xlsx?dl=0 Suppl file5_target expression https://www.dropbox.com/s/0rdd40n630u8zdy/Suppl%20file5_target%20expression.xlsx?dl=0


Ucp1

Gapdh

Sh-RBPMS2Sh-Ctl

Ucp1

Gapdh

Sh-Ctl Sh-AKPA1

020406080100 * *

*Maximal

0

5

10

15* *

ATP turnover

*

OC

R (p

Mol

/min

*ug)

Basal

010203040

**

Proton leak

0

10

20

30Control

sh-AKAP1

sh-RBPMS2

A B

C

0 20 40 60 80 1000

30

60

90

120 sh-RBPMS2sh-AKAP1

sh-Ctl

OC

R (p

Mol

/min

*ug)

Olig

omyc

in

FCC

P

Ret

enon

e

Figure S1


Ucp1

Pgc1α

Cidea

Cox7

Pparγ

0

1

2

Rel

ativ

e ex

pres

sion

Grsf10

1

2

Sh-CtlSh-Grsf1

Rel

ativ

e ex

pres

sion

Grsf1

0

1

2

Sh-CtlSh-Grsf1

Rel

ativ

e ex

pres

sion

* *

A

Larp

40

1

2 Sh-CtlSh-Larp4

Rel

ativ

e ex

pres

sion

Ucp1

Pgc1αCideaCox7Pparγ

0

1

2 Sh-CtlSh-Larp4

*

B

Figure S2

Knockdown of Grsf1 and Larp4

Fabp4

Adipoq

Cebpα

Pparα

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

Ybx2

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

**

E

pMKO-shRNA, BAT

Sh-Ctl Sh-1 Sh-2

Ybx2

0.0

0.5

1.0

1.5

Sh-Ctl(pMKO)Sh-1(pMKO)

Sh-3(pMKO)

Rela

tive

expr

essi

on

**

Fabp4

Adipoq

Cebpa

Pparγ

0.0

0.5

1.0

1.5 ShRNA-ControlShRNA-1ShRNA-3

Sh-Ctl (pMKO) Sh-1 (pMKO) Sh-2 (pMKO)

Ucp1

Cidea

Pparα Di

o2

Prdm16

Cox4

Cox7

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

Ybx2

Fabp4

Adipoq

Pparγ

Ucp1

Pgc1α

Cidea

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e ex

pres

sion

* ** * * *

* **

** *

ShRNA-ControlShRNA-1ShRNA-3


H I J

L

K

C D

F G

*

Ucp1

Cidea

Pgc1α

Pparα

Dio2

Prdm16

Cox7

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

** *

** *

G

pMKO-shRNA, WAT

Sh-CtlSh-3

Sh-CtlSh-1

** ** **** *

** **

* ShRNA-ControlShRNA-1ShRNA-3


sh-Ctlsh-1sh-2

0.0

0.5

1.0

1.5

TAG/protein


WT KO85

90

95

100

Fat m

ass

(%BW

)

WT KO1.2

1.4

1.6

1.8

2.0

Fat m

ass

(%BW

)

WT KO1.0

1.2

1.4

1.6

1.8

Fat m

ass

(%BW

)

YBX2

Pgc1α

Pparα

Prdm16

Ucp1

Pparγ2

Hoxc10

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e ex

pres

sion

Adipoq

Cebpα

Pparγ2

Fabp4

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

A B

Figure S3

Lean iWAT eWATC D

WTKO

E BAT F

G

H

iWAT

Glucose tolerance test

0 30 60 90 1200

100

200

300

400 WTKO

Time (min)

Glu

cose

(mg/

dl)

**

Ucp1

Prdm16

Pparα

Pgc1α Dio2

Cidea

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

* * *

BAT

WT KO4

5

6

7

Fat m

ass

(%BW

)

Fat

0 30 60 90 1200

50

100

150

Time (min)

Glu

cose

(%)

Insulin tolerance testWTKO


Adipoq

Cebpα

Pparγ2

0.0

0.5

1.0

1.5

KO

WTEn

richm

ent s

core

Adipogenesis0.00

-0.15

-0.30

-0.45

NES: -1.9P-value: 0.0

Oxidative Phosphorylation0.00

-0.20

-0.40

-0.60

NES= -2.2P-value<10-5

Enric

hmen

t sco

re

Fatty acid oxidation


0.00

-0.20

-0.40

-0.60

Cellular respiration


0.00

-0.20

-0.40

-0.60

Ybx2

0.0

0.5

1.0

1.5

Ucp1

Pgc1α

Prdm16

Cidea

Cox8

0.0

0.5

1.0

1.5

KOWT

Rel

ativ

e Ex

pres

sion

Rel

ativ

e Ex

pres

sion

Figure S4

** * * *

**

A B

C

D

Rank (KO/WT)

Rank (KO/WT) Rank (KO/WT)

Rank (KO/WT)

*


Insulin - - ++++ - - ++++WT KO WT WT KO KO WT KO WT WT KO KO

Cold

P-AKT

AKT

RT

Figure S5

0 50 100 150 2000246810

minutes

OD/weight(g)

WTKO

CB

0 5 10 15 20050100150200250

05101520

minutes

O2(nmol/ml)

pmol/(s*ug)WT

FCCP

AntiA

0 5 10 15 20050100150200250

0

5

10

15

20

minutes

pmol/(s*ug)KO

O2 concentration

O2 consumption rate

FCCP

AntiA

Basal

Fccp

05101520

pmol/(s*µg)

WTKO

*

A

Ybx2

Ucp1

Pgc1α

Pparα

Prdm16

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

*

D


Figure S6

WT

WT

WT

WT-Cold

WT-Cold

WT-Cold KO KO KO

KO-Cold

KO-Cold

KO-Cold

0

2000

4000

6000

8000

FPK

M

Ucp1

WT

WT

WT

WT-Cold

WT-Cold

WT-Cold KO KO KO

KO-Cold

KO-Cold

KO-Cold

0

100

200

300

400

FPK

M

Pgc1α

WT

WT

WT

WT-Cold

WT-Cold

WT-Cold KO KO KO

KO-Cold

KO-Cold

KO-Cold

0

50

100

150

200

250

FPK

M

Dio2

WT

WT

WT

WT-Cold

WT-Cold

WT-Cold KO KO KO

KO-Cold

KO-Cold

KO-Cold

0

50

100

150

Room Temp Cold 6 hrs Room Temp Cold 6 hrs

Wild type knockout

FPK

M

Elovl3

Dow

n-re

gula

ted

GO

0 1 2 3 4 5 10 15

Lipid biosynthetic process

Phospholipidbiosyntheticprocess

Triglyceridemetabolicprocess

Mitochondrion

Fattyacidmetabolicprocess

-log2(P-value)0 2 4 6 8 10

External side of plasma membrane

Tube development

Up-

regu

late

d G

O

Polysaccharide binding

Vasculature development

Cell-cell adhesion

-log2(P-value)

A

B

C

Room Temp Cold 6 hrs Room Temp Cold 6 hrs

Wild type knockout

Dgat2

Dio2

Agpat2

Slc2a4

PGc1a

Acaca

Elovl3

Fasn

2 -2 0

WTCOLD

WTRT

KOCOLD

KORT

Log2(fold change)


Ucp1

Pgc1α

Pparα

Prdm16

Elovl3

Dio2Cide

a0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

WTKO

** * *

**

Mogat1

Acaca

Srebf1

Dgat1

Fasn

Elovl6

Dgat2

Scd1

Glut4

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

** *

** *

Figure S7

A B

WT KO

0.0

0.5

1.0

1.5

2.0

iWAT

mas

s (%

BW

)

iWAT

BAT

mas

s (%

BW

)

WT KO

0.0

0.2

0.4

0.6

0.8

BAT

*

WT KO

0

10

20

30

40

Body

wei

ght(g

)

BWC

D

E

WTKO


Figure S8

0 2 4 6 840

60

80

100

120

Rem

aini

ng p

erce

ntag

e

Hours

Ybx2+WT

Ybx2+Mutant

Vector+WT

Vector+Mutant

Vector+WT T1/2 = 4.797 hoursVector+Mutant T1/2 = 4.915 hours

Ybx2+Mutant T1/2 = 4.105 hoursYbx2+WT T1/2 = 8.385 hours

A

B

C

IgG

Anti-Ybx2

0

20

40

60

80

IP/Input

NEBasal


Ybx2

Serca

1

Serca

2

Serca

3

Sarco

lipin

0

2

4

6

8

Rel

ativ

e E

xpre

ssio

n WT

KO *

Basal

Maxim

alATP

Uncouplin

g0

50

100

150

pm

ol/(s

*mg)

WT

KO

WT

KO

A

B

Ybx2Nuclear Nuclearcytosol cytosol

Gapdh

RT 4oCC

Figure S9


Q-PCR Primers sets for mouse genes

Name

ucp1

Prdm16

pgc1α

Cidea

Dio2

Elovl3

ppar-α

AdipoQ

CEBPα

Fabp4

pparg2

Cox7a1

Cox8b

Ybx2

Rbpms2

Akap1

Grsf1

Larp4

Mogat1

Acaca

Srebf1

Dgat1

Fasn

Elovl6

Dgat2

Scd1

Glut4

ATP2A1

ATP2A2

ATP2A3

SLN


Q-PCR Primers sets for Human genes

hYBX2

hRBPMS2

hGRSF1

hAKAP1

hLARP4

YBX2 cloning primers in XZ201

YBX2 F1

YBX2 R1

Pgc1a cloning primers for XZ201

mPGC1a-F

mPGC1a-R

Oligo for MSCV-pgkGFP-U3-U6P-Bbs vector

shRNA control

AKAP1_Sh1

AKAP1_Sh2

AKAP1_sh3

Rbpms2_sh1

Rbpms2_sh2

Rbpms2_sh3

Ybx2_sh1

Ybx2_sh2

Ybx2_sh3

Oligo for pMKO shRNA plasmids

sh-Ctl_top

sh-Ctl_bottom

Ybx2-sh1_top

Ybx2-sh1_bottom

Ybx2-sh2_top

Ybx2-sh2_bottom

Oligo for pSUPER.GFP.NEO shRNA plasmids

mPGC1a-sh-T2


mPGC1a-sh-B2

PCR primers used to segments of Pgc1a's 3'UTR

T7-Pgc1a-Seq1-F

T7-Pgc1a-Seq1-R

T7-Pgc1a-Seq2-F

T7-Pgc1a-Seq2-R

T7-Pgc1a-Seq3-F

T7-Pgc1a-Seq3-R

T7-Pgc1a-Seq4-F

T7-Pgc1a-Seq4-R

T7-Pgc1a-Seq3.1-F

T7-Pgc1a-Seq3.1-R

T7-Pgc1a-Seq3.2-F

T7-Pgc1a-Seq3.2-R

T7-Pgc1a-Seq3.3-F

T7-Pgc1a-Seq3.3-R

T7-Pgc1a-Seq3.4-F

T7-Pgc1a-Seq3.4-R

T7-Pgc1a-Seq3.5-F

T7-Pgc1a-Seq3.5-R

T7-Pgc1a-Seq3.6-F

T7-Pgc1a-Seq3.6-R

T7-Pgc1a-Seq3.7-F

T7-Pgc1a-Seq3.7-R

T7-Pgc1a-Seq3.8-F

T7-Pgc1a-Seq3.8-R

Pgc1aFrag_psicheck2_F-XhoI

Pgc1aFrag_psicheck2_R2_WT

Pgc1aFrag_psicheck2_R1_mutant


Forward primers

ACTGCCACACCTCCAGTCATT

CAGCACGGTGAAGCCATTC

CCCTGCCATTGTTAAGACC

TGCTCTTCTGTATCGCCCAGT

CAGTGTGGTGCACGTCTCCAATC

TCCGCGTTCTCATGTAGGTCT

AGAGCCCCATCTGTCCTCTC

CGATTGTCAGTGGATCTGACG

TGCGCAAGAGCCGAGATAAA

ACAAGCTGGTGGTGGAATGTG

GCATGGTGCCTTCGCTGA

CAGCGTCATGGTCAGTCTGT

GAACCATGAAGCCAACGACT

TGGGCACAGTCAAATGGTTC

TCCATTCAAGGGCTATGAAGGG

ACATTTTCCCCCAACACAGC

TTGCCTTTCCAAGCCAATGC

ATGCTGAAGTGTGCCAGAAG

TGGTGCCAGTTTGGTTCCAG

GATGAACCATCTCCGTTGGC

TGACCCGGCTATTCCGTGA

GTGCCATCGTCTGCAAGATTC

GGAGGTGGTGATAGCCGGTAT

GAAAAGCAGTTCAACGAGAACG

GCGCTACTTCCGAGACTACTT

TTCTTGCGATACACTCTGGTGC

CTGTCGCTGGTTTCTCCAACT

TGTTTGTCCTATTTCGGGGTG

GAGAACGCTCACACAAAGACC

CGTCGCTTCTCGGTGACAG

AGAGACTGAGGTCCTTGGTA


GATTCATCAACAGGAATGA

CCTGATCAAGCTCACTGCAA

GCCAGCGGTATGTGGAAGTAT

TGTCTCGGGAGCATGTCTTG

AAAGTGAGACCAAGTCATAAGCG

AAACTCGAGATGAGCGAGGCGGAGGCGT

AAAGAATTCGGAGGGGGATGCTGGGTAG

GACTCGAGatggcttgggacatgTGCAGCCAAGACTCTGTAT

GAGTTAACttacctgcgcaagcttctct

AAAACAACAAGATGAAGAGCACCAAGTCGACTTGGTGCTCTTCATCTTGTTG

AAAAGGAAGTTGCCGAGTAGCTTTGGTCGACCAAAGCTACTCGGCAACTTCC

AAAAGGCAAATTAGGTCTGACTTTGGTCGACCAAAGTCAGACCTAATTTGCC


AAAAGCAAATGTGTGTATGGTTTGTGTCGACACAAACCATACACACATTTGC

AAAAGCGACACCAAATCCCACCAGTGTCGACACTGGTGGGATTTGGTGTCGC

AAAAGCATTGAATGGTATTCGCTTTGTCGACAAAGCGAATACCATTCAATGC

AAAAGGTGATCAACAGCAGGGAGATGTCGACATCTCCCTGCTGTTGATCACC

AAAAGTCCACGAAACCGTCCCTACTGTCGACAGTAGGGACGGTTTCGTGGAC

AAAAGGAATGGTTACGGATTCATCAGTCGACTGATGAATCCGTAACCATTCC

CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG

AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG

CCGGACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGTTTTTG

AATTCAAAAACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGT

CCGGGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACCTTTTTG

AATTCAAAAAGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACC

Oligo for pSUPER.GFP.NEO shRNA plasmids

GATCCCCGCAACATGCTCAAGCCAAACCCGAAGGTTTGGCTTGAGCATGTTGCTTTTTA


AGCTTAAAAAGCAACATGCTCAAGCCAAACCTTCGGGTTTGGCTTGAGCATGTTGCGGG

PCR primers used to segments of Pgc1a's 3'UTR

GATCTAATACGACTCACTATAGGTGTTCCCAGGCTGAGGAAT

CAGGACAAAGGACAAACTAC

GATCTAATACGACTCACTATAGGAAGTTTCTGTAGTTTGTCC

TCTTCAGACACACATTGACT

GATCTAATACGACTCACTATAGCTTTGAAGCCAGTATCTCTT

CAAGACAACGTATGTTTTTAAAGTTGG

GATCTAATACGACTCACTATAGCCCTGGATCATGGACATGA

AGGCTGATGTGTACTGCACA


CCTGATGCTCAAAATGGAG

GATCTAATACGACTCACTATAGATGGTGTTGTTCTTGGTGAC

GTAAGATAGTGTTGGGTGAGAGAG

GATCTAATACGACTCACTATAGGCATTTACTGTTTGGCTGAC

CCTGCATTTATCCTACAGAACAAG

GATCTAATACGACTCACTATAGGTTCACAGGTTCTGCGTTAC



AACACCATGGTCGTATCAGA

GATCTAATACGACTCACTATAGTCGTTTGGGAAACTCAGCTCTC

TCCAGAAAATTCATGTCAGC

GATCTAATACGACTCACTATAGGGAGTCACTAAACTTTGGAG

CGCAGAACCTGTGAACACAA

GATCTAATACGACTCACTATAGGTCGAATGCTTGCTCAAGTG


TTTctcgaggccattgaatctgggtgg

TTTgcggccgcctttagtttggcgttcacaaaga

TTTgcggccgcaagatagtcttcagacacacattgact


Reverse primers

CTTTGCCTCACTCAGGATTGG

GCGTGCATCCGCTTGTG

TGCTGCTGTTCCTGTTTTC

GCCGTGTTAAGGAATCTGCTG

TGAACCAAAGTTGACCACCAG

GGACCTGATGCAACCCTATGA

ACTGGTAGTCTGCAAAACCAAA

CAACAGTAGCATCCTGAGCCCT

CCTTCTGTTGCGTCTCCACG

CCTTTGGCTCATGCCCTTT

TGGCATCTCTGTGTCAACCATG

AGAAAACCGTGTGGCAGAGA

GCGAAGTTCACAGTGGTTCC

AACACTCCGCAGAAACTTCC

ATGCGTTTTTGGCTGCTTCC

AGCAGTGGAAAGGTGTAAGC

TCAAAGTGCACGTCAGCTTC

TTCTCATGCGGCTTCTCAAC

TGCTCTGAGGTCGGGTTCA

GACCCAATTATGAATCGGGAGTG

CTGGGCTGAGCAATACAGTTC

GCATCACCACACACCAATTCAG

TGGGTAATCCATAGAGCCCAG

AGATGCCGACCACCAAAGATA

GGGCCTTATGCCAGGAAACT

CGGGATTGAATGTTCTTGTCGT

CCCATAGCATCCGCAACATA

AATCCGCACAAGCAGGTCTTC

CAATTCGTTGGAGCCCCAT

AAGAGGTCCTCAAACTGCTCC

GGTGATGAGGACAACTGTGA


CCAGTTACATTAGTGGCTTC

CTCTTGGCCATCTTGGTGTT

AGGCGAAGATTTGACCTGCAA

GCCGACTCGATGAACCTACTT

ACCAGTTGCTATTGTGTGCAAA



AAAAGCGACACCAAATCCCACCAGTGTCGACACTGGTGGGATTTGGTGTCGC

AAAAGGTGATCAACAGCAGGGAGATGTCGACATCTCCCTGCTGTTGATCACC

AAAAGTCCACGAAACCGTCCCTACTGTCGACAGTAGGGACGGTTTCGTGGAC

CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG

AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG

CCGGACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGTTTTTG

AATTCAAAAACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGT

CCGGGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACCTTTTTG

AATTCAAAAAGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACC

GATCCCCGCAACATGCTCAAGCCAAACCCGAAGGTTTGGCTTGAGCATGTTGCTTTTTA


AGCTTAAAAAGCAACATGCTCAAGCCAAACCTTCGGGTTTGGCTTGAGCATGTTGCGGG


GACCCAATTATGAATCGGGAGTG


RNA Binding Protein, Ybx2, Regulates RNA Stability …...RNA binding protein, Ybx2, regulates RNA stability during cold-induced brown fat activation Dan Xu1,2*, Shaohai Xu3, Aung Maung

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