Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae.
Project description:Alternative polyadenylation gives rise to a wide variety of mRNA isoforms with distinct 3' ends; an individual gene can yield many 3' mRNA isoforms and has a typical pattern of poly(A) site use. To identify possible determinants of polyadenylation site distribution, we have used CRISPR to create Saccharomyces cerevisiae strains harboring precise ORF deletions in candidate genes encoding various factors involved in gene expression. We have performed 3' READS to determine the genome-wide pattern of 3' mRNA isoform endpoints in these mutant strains.
