期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：E3374-E3383
DOI：10.1073/pnas.1418603112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMultiple studies highlight the role of various proteins in regulation of alternative splicing; however, the regulatory role of distinct posttranslational modifications during alternative splicing that contribute to tumorigenesis is enigmatic. Here we report a previously unidentified noncanonical mechanism of regulation of alternative splicing modulated by deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa) via Scaffold/matrix-associated region-binding protein 1 (SMAR1)-histone deacetylase 6 (HDAC6) complex. SMAR1 in complex with HDAC6 maintains Sam68 in a deacetylated state. We observed that ERK-1/2-dependent phosphorylation of SMAR1, knockdown of SMAR1, or loss of heterozygosity facilitates CD44 variant exon inclusion via Sam68 acetylation and thus confers invasive and metastatic potential in breast tumor cells. Our findings provide key insights into regulation of alternative splicing and the potential for therapeutic intervention during tumor metastasis. 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.
关键词：SMAR1 ; Sam68 ; HDAC6 ; alternative splicing ; deacetylation
