ABSTRACT

Recently, by combining transcriptomics with functional splicing reporter assays we were able to identify GT > GC > TT as the three highest ranked dinucleotides of human 5ʹ splice sites (5’ss). Here, we have extended our investigations to the proteomic characterization of nuclear proteins that bind to canonical and noncanonical 5’ss. Surprisingly, we found that U1 snRNP binding to functional 5’ss sequences prevented components of the DNA damage response (DDR) from binding to the RNA, suggesting a close link between spliceosome arrangement and genome stability.

We demonstrate that all tested noncanonical 5’ss sequences are bona-fide targets of the U2-type spliceosome and are bound by U1 snRNP, including U1-C, in the presence of splicing enhancers. The quantity of precipitated U1-C protein was similar for all noncanonical 5’ss dinucleotides, so that the highly different 5’ss usage was likely due to a later step after early U1 snRNP binding.

In addition, we show that an internal GT at positions +5/+6 can be advantageous for splicing at position +1 of noncanonical splice sites. Likewise, and in agreement with previous observations, splicing inactive U1 snRNP binding sites could serve as splicing enhancers, which may also explain the higher abundance of U1 snRNPs compared to other U snRNPs. Finally, we observe that an arginine-serine (RS)-rich domain recruitment to stem loop I of the U1 snRNA is functionally sufficient to promote exon-definition and upstream 3’ss activation.

摘要

最近，通过将转录组学与功能性剪接报告基因分析相结合，我们能够将 GT > GC > TT 鉴定为人类 5ʹ 剪接位点 (5's) 中排名最高的三个二核苷酸。在这里，我们将我们的研究扩展到结合规范和非规范 5 ss 的核蛋白的蛋白质组学特征。令人惊讶的是，我们发现 U1 snRNP 与功能性 5's 序列结合阻止了 DNA 损伤反应 (DDR) 的成分与 RNA 结合，表明剪接体排列和基因组稳定性之间存在密切联系。

我们证明所有经过测试的非规范 5 ss 序列都是 U2 型剪接体的真正目标，并且在剪接增强剂存在的情况下受 U1 snRNP（包括 U1-C）的约束。所有非规范 5'ss 二核苷酸的 U1-C 蛋白沉淀量相似，因此 5's 的使用差异很大可能是由于早期 U1 snRNP 结合后的后续步骤。

此外，我们表明，+5/+6 位的内部 GT 有利于非规范剪接位点 +1 位的剪接。同样，与之前的观察结果一致，剪接无活性的 U1 snRNP 结合位点可以作为剪接增强剂，这也可以解释与其他 U snRNP 相比 U1 snRNP 的丰度更高。最后，我们观察到富含精氨酸丝氨酸 (RS) 的域募集到 U1 snRNA 的茎环 I 在功能上足以促进外显子定义和上游 3 ss 激活。