Nakka Kiran Kumar, Chaudhary Nidhi, Joshi Shruti, Bhat Jyotsna, Singh Kulwant, Chatterjee Subhrangsu, Malhotra Renu, De Abhijit, Santra Manas Kumar, Dilworth F Jeffrey, Chattopadhyay Samit
Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India; Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada;
Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India;
Proc Natl Acad Sci U S A. 2015 Jun 30;112(26):E3374-83. doi: 10.1073/pnas.1418603112. Epub 2015 Jun 15.
Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.
前体信使核糖核酸（pre-mRNA）剪接是一个复杂的调控枢纽，受多种反式作用因子及其翻译后修饰的调节，通过可变剪接产生动态转录组。已知反式作用因子的信号诱导磷酸化和去磷酸化可调节可变剪接。然而，其他翻译后修饰，如去乙酰化/乙酰化、甲基化和泛素化，以信号依赖或非依赖方式调节可变剪接的作用仍不清楚。在此，我们证明支架/基质附着区域结合蛋白1（SMAR1）通过组蛋白去乙酰化酶6（HDAC6）介导的RNA结合蛋白Sam68（有丝分裂时68 kDa的Src相关底物）去乙酰化来负向调节可变剪接。SMAR1在核剪接斑点中富集，并与参与剪接位点识别的小核RNA（snRNA）相关联。调节可变剪接的细胞外信号调节激酶-丝裂原活化蛋白激酶（ERK-MAPK）途径促进ERK-1/2介导SMAR1在苏氨酸345和360处的磷酸化，并将SMAR1定位到细胞质中，阻止其与Sam68相互作用。我们发现，内源性地，SMAR1通过HDAC6使Sam68保持去乙酰化状态。然而，SMAR1的敲低或ERK介导的磷酸化会释放抑制性的SMAR1-HDAC6-Sam68复合物，促进Sam68乙酰化和可变剪接。此外，乳腺癌细胞中16号染色体q24.3位点的杂合性缺失（人类SMAR1同源物BANP已定位在此位点）会增强Sam68乙酰化和CD44可变外显子的包含。此外，对缺乏SMAR1的人乳腺癌MCF-7细胞进行小鼠尾静脉注射，结果显示CD44可变外显子的包含增加以及随之而来的转移倾向，证实了SMAR1在调节可变剪接中的功能作用。因此，我们的结果揭示了SMAR1通过Sam68去乙酰化介导可变剪接的信号依赖和非依赖调节的复杂分子机制。
