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

Erkelenz, Steffen ORCID: 0000-0003-4763-1240, Poschmann, Gereon, Ptok, Johannes, Mueller, Lisa and Schaal, Heiner ORCID: 0000-0002-1636-4365 . Profiling of cis- and trans-acting factors supporting noncanonical splice site activation. RNA Biol.. PHILADELPHIA: TAYLOR & FRANCIS INC. ISSN 1555-8584

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DOI:10.1080/15476286.2020.1798111

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 5MODIFIER LETTER PRIME 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.

Item Type:

Journal Article

Creators:

Creators	Email	ORCID	ORCID Put Code
Erkelenz, Steffen	UNSPECIFIED	orcid.org/0000-0003-4763-1240	UNSPECIFIED
Poschmann, Gereon	UNSPECIFIED	UNSPECIFIED	UNSPECIFIED
Ptok, Johannes	UNSPECIFIED	UNSPECIFIED	UNSPECIFIED
Mueller, Lisa	UNSPECIFIED	UNSPECIFIED	UNSPECIFIED
Schaal, Heiner	UNSPECIFIED	orcid.org/0000-0002-1636-4365	UNSPECIFIED