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.
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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
Page 3 of 51 Diabetes
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)
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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
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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
Page 6 of 51Diabetes
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
Page 7 of 51 Diabetes
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-
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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
Page 9 of 51 Diabetes
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
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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
Page 11 of 51 Diabetes
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
Page 12 of 51Diabetes
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.
Page 13 of 51 Diabetes
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
Page 14 of 51Diabetes
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
Page 15 of 51 Diabetes
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.)
Page 16 of 51Diabetes
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.
Page 17 of 51 Diabetes
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A B C D
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Page 22 of 51Diabetes
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Cebpα
Pparγ2
0.0
0.5
1.0
1.5
Rbpms2
0.0
0.5
1.0
1.5 ** **
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Knockdown Pan-adipocyte markers
BAT-selective genes
D
0 2 4 6 0 2 4 6 BAT WAT
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
Page 23 of 51 Diabetes
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
FAO and UCP1 Western blot in overexpression cells
MAcd
B actin
UCP-1
CPT1AFAO and UCP1 Western blot in overexpression cells
MAcd
B actin
UCP-1
CPT1A
FAO and UCP1 Western blot in overexpression cells
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
Page 24 of 51Diabetes
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
)
Page 25 of 51 Diabetes
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
Page 26 of 51Diabetes
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
Page 27 of 51 Diabetes
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
*
*
Page 28 of 51Diabetes
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.
Page 29 of 51 Diabetes
(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
Page 30 of 51Diabetes
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.
Page 31 of 51 Diabetes
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
Page 32 of 51Diabetes
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
Page 33 of 51 Diabetes
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
Sh-Ctl (pMKO) Sh-1 (pMKO) Sh-2 (pMKO)
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-Ctl (pMKO) Sh-1 (pMKO) Sh-2 (pMKO)
sh-Ctlsh-1sh-2
0.0
0.5
1.0
1.5
TAG/protein
Page 34 of 51Diabetes
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
Page 35 of 51 Diabetes
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
NES= -1.7P-value<10-5
0.00
-0.20
-0.40
-0.60
Cellular respiration
NES= -1.8P-value<10-5
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)
*
Page 36 of 51Diabetes
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
Page 37 of 51 Diabetes
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)
Page 38 of 51Diabetes
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
Page 39 of 51 Diabetes
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
Page 40 of 51Diabetes
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
Page 41 of 51 Diabetes
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
Page 42 of 51Diabetes
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
Page 43 of 51 Diabetes
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
Page 44 of 51Diabetes
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
Page 45 of 51 Diabetes
GATTCATCAACAGGAATGA
CCTGATCAAGCTCACTGCAA
GCCAGCGGTATGTGGAAGTAT
TGTCTCGGGAGCATGTCTTG
AAAGTGAGACCAAGTCATAAGCG
AAACTCGAGATGAGCGAGGCGGAGGCGT
AAAGAATTCGGAGGGGGATGCTGGGTAG
GACTCGAGatggcttgggacatgTGCAGCCAAGACTCTGTAT
GAGTTAACttacctgcgcaagcttctct
AAAACAACAAGATGAAGAGCACCAAGTCGACTTGGTGCTCTTCATCTTGTTG
AAAAGGAAGTTGCCGAGTAGCTTTGGTCGACCAAAGCTACTCGGCAACTTCC
AAAAGGCAAATTAGGTCTGACTTTGGTCGACCAAAGTCAGACCTAATTTGCC
AAAAGGAAGTTGCCGAGTAGCTTTGGTCGACCAAAGCTACTCGGCAACTTCC
AAAAGCAAATGTGTGTATGGTTTGTGTCGACACAAACCATACACACATTTGC
AAAAGCGACACCAAATCCCACCAGTGTCGACACTGGTGGGATTTGGTGTCGC
AAAAGCATTGAATGGTATTCGCTTTGTCGACAAAGCGAATACCATTCAATGC
AAAAGGTGATCAACAGCAGGGAGATGTCGACATCTCCCTGCTGTTGATCACC
AAAAGTCCACGAAACCGTCCCTACTGTCGACAGTAGGGACGGTTTCGTGGAC
AAAAGGAATGGTTACGGATTCATCAGTCGACTGATGAATCCGTAACCATTCC
CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG
AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG
CCGGACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGTTTTTG
AATTCAAAAACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGT
CCGGGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACCTTTTTG
AATTCAAAAAGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACC
Oligo for pSUPER.GFP.NEO shRNA plasmids
GATCCCCGCAACATGCTCAAGCCAAACCCGAAGGTTTGGCTTGAGCATGTTGCTTTTTA
Page 46 of 51Diabetes
AGCTTAAAAAGCAACATGCTCAAGCCAAACCTTCGGGTTTGGCTTGAGCATGTTGCGGG
PCR primers used to segments of Pgc1a's 3'UTR
GATCTAATACGACTCACTATAGGTGTTCCCAGGCTGAGGAAT
CAGGACAAAGGACAAACTAC
GATCTAATACGACTCACTATAGGAAGTTTCTGTAGTTTGTCC
TCTTCAGACACACATTGACT
GATCTAATACGACTCACTATAGCTTTGAAGCCAGTATCTCTT
CAAGACAACGTATGTTTTTAAAGTTGG
GATCTAATACGACTCACTATAGCCCTGGATCATGGACATGA
AGGCTGATGTGTACTGCACA
GATCTAATACGACTCACTATAGCTTTGAAGCCAGTATCTCTT
CCTGATGCTCAAAATGGAG
GATCTAATACGACTCACTATAGATGGTGTTGTTCTTGGTGAC
GTAAGATAGTGTTGGGTGAGAGAG
GATCTAATACGACTCACTATAGGCATTTACTGTTTGGCTGAC
CCTGCATTTATCCTACAGAACAAG
GATCTAATACGACTCACTATAGGTTCACAGGTTCTGCGTTAC
CAAGACAACGTATGTTTTTAAAGTTGG
GATCTAATACGACTCACTATAGCTTTGAAGCCAGTATCTCTT
AACACCATGGTCGTATCAGA
GATCTAATACGACTCACTATAGTCGTTTGGGAAACTCAGCTCTC
TCCAGAAAATTCATGTCAGC
GATCTAATACGACTCACTATAGGGAGTCACTAAACTTTGGAG
CGCAGAACCTGTGAACACAA
GATCTAATACGACTCACTATAGGTCGAATGCTTGCTCAAGTG
CAAGACAACGTATGTTTTTAAAGTTGG
TTTctcgaggccattgaatctgggtgg
TTTgcggccgcctttagtttggcgttcacaaaga
TTTgcggccgcaagatagtcttcagacacacattgact
Page 47 of 51 Diabetes
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
Page 48 of 51Diabetes
CCAGTTACATTAGTGGCTTC
CTCTTGGCCATCTTGGTGTT
AGGCGAAGATTTGACCTGCAA
GCCGACTCGATGAACCTACTT
ACCAGTTGCTATTGTGTGCAAA
AAAAGGAAGTTGCCGAGTAGCTTTGGTCGACCAAAGCTACTCGGCAACTTCC
AAAAGGAAGTTGCCGAGTAGCTTTGGTCGACCAAAGCTACTCGGCAACTTCC
AAAAGCGACACCAAATCCCACCAGTGTCGACACTGGTGGGATTTGGTGTCGC
AAAAGGTGATCAACAGCAGGGAGATGTCGACATCTCCCTGCTGTTGATCACC
AAAAGTCCACGAAACCGTCCCTACTGTCGACAGTAGGGACGGTTTCGTGGAC
CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG
AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG
CCGGACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGTTTTTG
AATTCAAAAACCCAAGGAGACAGCACCATTCTCGAGAATGGTGCTGTCTCCTTGGGT
CCGGGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACCTTTTTG
AATTCAAAAAGGTGATCAACAGCAGGGAGATCTCGAGATCTCCCTGCTGTTGATCACC
GATCCCCGCAACATGCTCAAGCCAAACCCGAAGGTTTGGCTTGAGCATGTTGCTTTTTA
Page 49 of 51 Diabetes
AGCTTAAAAAGCAACATGCTCAAGCCAAACCTTCGGGTTTGGCTTGAGCATGTTGCGGG
Page 50 of 51Diabetes
GACCCAATTATGAATCGGGAGTG
Page 51 of 51 Diabetes