SRSF1抑制DOK7内含子4的内含子5'剪接位点的选择，以生成功能性全长Dok-7蛋白。,Scientific Reports

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SRSF1抑制DOK7内含子4的内含子5'剪接位点的选择，以生成功能性全长Dok-7蛋白。
Scientific Reports ( IF 3.8 ) Pub Date : 2017-Sep-05 , DOI: 10.1038/s41598-017-11036-z
Khalid Bin Ahsan , Akio Masuda , Mohammad Alinoor Rahman , Jun-ichi Takeda , Mohammad Nazim , Bisei Ohkawara , Mikako Ito , Kinji Ohno

Dok-7是一种非催化衔接蛋白，可促进神经肌肉接头处的凝集素诱导的乙酰胆碱受体（AChR）聚集。DOK7内含子4的5'剪接位点（SS）的替代选择会生成规范的和移框的转录本。我们发现规范的全长Dok-7增强了AChR聚类，而截短的Dok-7却没有。我们通过块扫描诱变鉴定了一个接近外显子4'3'末端的剪接顺式元件。RNA亲和纯化和质谱分析表明SRSF1与顺式元件结合。敲除SRSF1可以增强DOK7内含子4的5'SS内含子的选择，而MS2介导的SRSF1与已确定的顺式元件的人工束缚可以抑制它。早期剪接体复合物的分离显示，SRSF1抑制了U1 snRNP与内含子5'SS的缔合，而是增强了U1 snRNP与内含子5'SS的缔合，这导致了经典DOK7转录本的上调。对CLIP-seq和RNA-seq的整体全局分析还表明，SRSF1直接与两个竞争性5'SS的上游结合会抑制数百种人类基因中内含子5'SS的选择。我们证明，SRSF1可以通过调节U1 snRNP的结合来严格调节相邻5'SS的替代选择。对CLIP-seq和RNA-seq的整体全局分析还表明，SRSF1直接与两个竞争性5'SS的上游结合会抑制数百种人类基因中内含子5'SS的选择。我们证明，SRSF1可以通过调节U1 snRNP的结合来严格调节相邻5'SS的替代选择。对CLIP-seq和RNA-seq的整体全局分析还表明，SRSF1直接与两个竞争性5'SS的上游结合会抑制数百种人类基因中内含子5'SS的选择。我们证明，SRSF1可以通过调节U1 snRNP的结合来严格调节相邻5'SS的替代选择。

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SRSF1 suppresses selection of intron-distal 5' splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein.

Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5' splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3' end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5' SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5' SS, and rather enhanced association of U1 snRNP to the intron-proximal 5' SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5' SSs suppresses selection of the intron-distal 5' SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5' SSs by modulating binding of U1 snRNP.

更新日期：2017-09-05

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