PAT-Seq: A method for simultaneous quantitation of gene expression, poly(A)-site selection and poly(A)-length distribution in yeast transcriptomes

Abstract

Next-generation sequencing (NGS) and its application to RNA (RNA-seq) has opened up multiple aspects of RNA processing to deep transcriptome-wide analysis at nucleotide resolution. This has been useful in delineating the transcribed areas of the genome, and in quantitation of RNA isoforms. Such isoforms can diversify the regulatory repertoire of mRNAs. For example, the 3′-end of mRNA can vary in two important ways, in the position chosen for cleavage and polyadenylation, and in the length of the poly(A)-tail. Accordingly, the step-up in resolution made possible by NGS has revealed an unexpectedly high level of alternative polyadenylation (APA). Moreover, it has massively simplified the transcriptome-wide detection of poly(A)-tail length changes. Here we present our approach to the study of 3′-end dynamics using a 3′-focused RNA-seq method called PAT-seq (for poly(A)-test sequencing). The approach records gene expression, APA, and poly(A)-tail changes between transcriptomes to reveal complex interplay between transcriptional and posttranscriptional control mechanisms. Show more