Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

TLDR

Alternative polyadenylation (APA) is a key post‑transcriptional regulatory mechanism, yet global profiling by microarrays is limited in coverage and quantitative accuracy. The authors developed PAS‑Seq, a deep‑sequencing method designed to quantitatively profile RNA polyadenylation across the transcriptome. PAS‑Seq accurately identifies poly(A) junctions in mRNAs and noncoding RNAs while providing quantitative abundance information for each polyadenylated transcript. PAS‑Seq analyses showed that 40–50 % of expressed genes exhibit APA, revealed evolutionarily conserved histone mRNA polyadenylation, uncovered novel mitochondrial RNA polyadenylation features, and demonstrated dynamic APA changes with 3′‑UTR lengthening during stem cell differentiation.

Abstract

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called P oly( A ) S ite Seq uencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.