Page 1 of 51 Diabetes 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.) Diabetes Publish Ahead of Print, published online September 29, 2017 Diabetes Page 2 of 51 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 3 of 51 Diabetes 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 Diabetes Page 4 of 51 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) Page 5 of 51 Diabetes 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