Impact on splicing in < em > Saccharomyces cerevisiae < /em > of random 50-base sequences inserted into an intron

RNA. 2023 Oct 25:rna.079752.123. doi: 10.1261/rna.079752.123. Online ahead of print.ABSTRACTIntron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing - 5' and 3' splice sites and a branch point - are especially well-characterized in Saccharomyces cerevisiae, but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in S. cerevisiae of random 50-nucleotide elements (N50) individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20%. N50 features, including higher GC content, presence of GU repeats and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases - such as the GU repeats - with other bases specifically within the reporter pre-mRNA analyzed. However, multiple models were unable to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex nonlinear interactions in RNA structures are not accurately captured by RNA structure prediction methods. Our results imply...
Source: RNA - Category: Genetics & Stem Cells Authors: Source Type: research