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

RNA Biology
Steffen ErkelenzHeiner Schaal

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...Continue Reading

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Citations

Nov 16, 2021·The EMBO Journal·Andrew M JobbinsIan C Eperon

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Datasets Mentioned

BETA
E-MTAB-4652

Methods Mentioned

BETA
PCR
transfections
transfection
in vitro transcription
pull down
pulldown
RNA-seq
fluorescence microscopy

Software Mentioned

ENSEMBL
Bioconductor
Perseus
R
HEXplorer
MaxQuant
BioMart
STAR aligner
rbamtools
CRAN

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