SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome.
SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome.
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DOI：
10.1038/srep13208
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发表时间：
2015-08-18
影响因子：
4.6
通讯作者：
Ohno K
中科院分区：
文献类型：
--
作者：
Rahman MA;Azuma Y;Nasrin F;Takeda J;Nazim M;Bin Ahsan K;Masuda A;Engel AG;Ohno K
乙酰胆碱酯酶（AChE）的催化亚基通过ColQ编码的胶原样尾亚基（ColQ）锚定在神经肌肉连接处的基底层。COLQ突变导致终板AChE缺乏。在终板AChE缺乏的患者中发现的预测COLQ 16外显子p.E415G的a -to- g突变导致16外显子的排他性跳变。RNA亲和纯化、质谱分析和sirna介导的基因敲除表明，该突变破坏了剪接增强RNA结合蛋白SRSF1的结合，并重新获得了剪接抑制RNA结合蛋白hnRNP H的结合。ms2介导的每个因子的人工拴系表明，SRSF1和hnRNP H通过仅结合外显子16中的靶标来拮抗地调节剪接。对人工突变体的进一步分析表明，SRSF1能够结合退行性结合基序，而hnRNP H则严格要求poly(G)的不间断延伸。突变破坏了下游内含子的剪接。早期剪接体复合物的分离表明，突变损害了U1-70K （snRNP70）与下游5 '剪接位点的结合。RNA-seq的全局剪接分析显示，在人类和小鼠的大脑中，携带hnRNP h结合的GGGGG基序的外显子比携带srsf1结合的GGAGG基序的外显子更容易被跳过。
The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5′ splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.