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

Authors :
Erkelenz S
Poschmann G
Ptok J
Müller L
Schaal H
Source :
RNA biology [RNA Biol] 2021 Jan; Vol. 18 (1), pp. 118-130. Date of Electronic Publication: 2020 Aug 05.
Publication Year :
2021

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 5' 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.

Details

Language :
English
ISSN :
1555-8584
Volume :
18
Issue :
1
Database :
MEDLINE
Journal :
RNA biology
Publication Type :
Academic Journal
Accession number :
32693676
Full Text :
https://doi.org/10.1080/15476286.2020.1798111