Abstract：
Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.
摘要：
平滑肌细胞（SMC）的分化取决于血清反应因子（SRF）及其共激活剂心肌素（MYOCD）。MYOCD在SMC基因转录程序中的作用已得到充分确立。相比之下,MYOCD在控制SMC特异性替代外显子使用中的作用，包括外显子剪接,没有被探索过。在目前的工作中，我们确定了四个剪接因子(MBNL1，RBPMS，RBPMS2和RBFOX2）与人SMC组织中的MYOCD相关。MYOCD家族成员在人冠状动脉SMCs中的强制表达在体外上调这些剪接因子的表达。对于转录本多样性的全球分析，我们在MYOCD转导后进行了RNA测序。我们用三种不同的方法分析了替代转录本。基于外显子的分析确定了1637个具有差异外显子使用的特征。例如,MYLK中编码telokin的3'外显子的使用相对于5'外显子增加，17kDatelokin与130kDaMYLK蛋白的比例也是如此。基于专用事件的分析确定了239个MYOCD驱动的剪接事件。涉及MBNL1、MCAM、ACTN1是最突出的，这通过变异特异性PCR分析得到证实。为了支持RBPMS和RBFOX2在MYOCD驱动的剪接中的作用，我们发现了它们在差异剪接的外显子周围的结合基序的富集。此外，MYOCD刺激的RBPMS或RBFOX2拮抗剪接事件的敲减，包括那些涉及ACTN1，VCL，MBNL1。支持MYOCD-SRF驱动剪接的体内作用，我们证明了在诱导型和SMC特异性Srf敲除小鼠中Rbpms表达和剪接的改变。我们得出的结论是MYOCD-SRF，部分通过RBPMS和RBFOX2诱导不同外显子使用和选择性剪接的程序，作为更广泛的SMC分化程序的一部分。
