RNA-Seq approach for accurate characterization of splicing efficiency of yeast introns

Publication date: Available online 26 March 2019Source: MethodsAuthor(s): Xuhua XiaAbstractIntrons in different genes, or even different introns within the same gene, often have different splice sites and differ in splicing efficiency (SE). One expects mass-transcribed genes to have introns with higher SE than weakly transcribed genes. However, such a simple expectation cannot be tested directly because variable SE for these genes is often not measured. Mechanistically, SE should depend on signal strength at key splice sites (SS) such as 5'SS, 3'SS and branchpoint site (BPS), i.e., SE = F(5'SS, 3'SS, BPS). However, without SE, we again cannot model how these splice sites contribute to SE. Here I present an RNA-Seq approach to quantify SE for each of the 304 introns in yeast (Saccharomyces cerevisiae) genes, including 24 in the 5'UTR, by measuring 1) number of reads mapped to exon-exon junctions (NEE) as a proxy for the abundance of spliced form, and 2) number of reads mapped to exon-intron junction (NEI5 and NEI3 at 5' and 3' ends of intron) as a proxy for the abundance of unspliced form. The total mRNA is NTotal = NEE + p*NEI5 + (1-p)*NEI3, with the simplest p = 0.5 but statistical methods were presented to estimate p from data. An estimated p is needed because NEI5 is expected to be smaller than NEI3 due to 1) step 1 splicing occurs before step 2 so EI5 is broken before EI3, 2) enrichment of poly(A) mRNA by oligo-dT, and 3) 5' degradation. SE is defined as the proportion (N...
Source: Methods - Category: Molecular Biology Source Type: research