Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors.
Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors.
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DOI：
10.1007/s00018-022-04497-7
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发表时间：
2022-08-01
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平滑肌细胞（SMC）的分化依赖于血清反应因子（SRF）及其共激活因子心肌素（MYOCD）。MYOCD在SMC基因转录程序中的作用已得到充分证实。相比之下，MYOCD在SMC特异性选择性外显子使用（包括外显子剪接）控制中的作用尚未研究。在目前的工作中，我们确定了四个剪接因子（MBNL1，RBPMS，RBPMS2和RBFOX 2），这些因子与人SMC组织中的MYOCD相关。在体外人冠状动脉平滑肌细胞中强制表达MYOCD家族成员可上调这些剪接因子的表达。对于转录物多样性的全局分析，我们在MYOCD转导后进行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，17 kDa telokin与130 kDa MYLK蛋白质的比例也是如此。专门的基于事件的分析确定了239个MYOCD驱动的剪接事件。涉及MBNL1、MCAM和ACTN1的事件是最突出的，这一点使用变异特异性PCR分析得到证实。为了支持RBPMS和RBFOX 2在MYOCD驱动的剪接中的作用，我们发现它们的结合基序在差异剪接的外显子周围富集。此外，RBPMS或RBFOX 2的敲低拮抗MYOCD刺激的剪接事件，包括涉及ACTN1、VCL和MBNL1的剪接事件。支持MYOCD-SRF驱动剪接的体内作用，我们证明了诱导型和SMC特异性Srf敲除小鼠中Rbpms表达和剪接的改变。我们得出结论，MYOCD-SRF，部分通过RBPMS和RBFOX 2，诱导程序的差异外显子使用和选择性剪接的一部分，更广泛的计划SMC分化。在线版本包含补充材料，可通过10.1007/s00018 - 022 - 04497 - 7获得。
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. The online version contains supplementary material available at 10.1007/s00018-022-04497-7.
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影响因子：
11.8
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