Profiling of cis- and trans-acting factors supporting noncanonical splice site activation

Recently, by combining transcriptomics with functional splicing reporter assays we were able to identify GT>GC>TT as the three highestranked dinucleotides of human 5ʹ splice sites (5’ss)(Erkelenz et al., 2018). Here, we have extended our investigations to theproteomic characterization of nuclear proteinsthat bindto 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 thatan arginine-serine (RS)-rich domain recruitment to stem loop I of the U1 snRNA is functionally sufficient to promoteexon-definition and upstream 3’ss activation.