Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

Abstract

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.

RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs

“…Alternatively spliced isoforms of the insulin receptor, lipin, nuclear receptor corepressor 1, and preadipocyte factor 1 (PREF-1) have all been associated with differentiation (49)(50)(51)(52). More recently, the alternative-splicing factor SAM68 was found to regulate adipogenesis by modulating intron 5 retention in mTOR transcripts and isoform switching of ribosomal S6 kinase (18,53). RNA-binding motif protein 4 (RBM4) has also been reported to regulate the splicing of adipocyte-specific genes, including the insulin receptor, PPARγ, and PREF-1, thereby facilitating brown adipocyte development (54).…”

Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

“…RNAi-mediated silencing of S6K1-p31 protein partially restored the differentiation of Sam68 −/− preadipocytes. Consistently, the presence of overexpressing S6K1-p31 variant mediated the differentiation defect in NIH3T3-L1 cells, which suggested the suppressive effect of S6K1-p31 variant on adipogenesis [ 44 ]. These findings indicate that Sam68 is required to prevent the expression of S6K1-p31 in adipocytes for adipogenesis to occur.…”

“…Downregulation of mTOR subsequently led to reduced phosphorylation of ribosomal protein S6 and Akt, which abrogated the effect of mTOR-mediated signaling on WAT development. Recently, Sam68 was demonstrated to modulate the splicing profile of ribosomal S6 kinase (S6K), which was involved in the mTOR signaling [ 44 ]. Ablation of Sam68 resulted in the generation of S6Kb1-002 transcript and the encoded S6K1-p31 protein, which is absent in the wild-type adipocytes.…”

Characterization of candidate genes for bovine adipogenesis reveals differences of TUSC5 isoforms caused by novel alternative splicing

“…For example, only the small soluble isoform of the delta-like non-canonical Notch ligand 1 (Dlk1) can inhibit adipocyte differentiation in mice [21]. Moreover, Sam68 was reported to regulate alternative splicing of the ribosomal protein S6 kinase (S6K1) during adipogenesis in mice [22]. In humans, alternative splicing events through the actions of the transformer 2 beta homolog (TRA2B), BCL2 associated athanogene 6 (BAG6), and mutS homolog 5 (MSH5) contribute to transcriptomic and proteomic diversity and could play important roles in regulating obesity [23].…”