title: An efficient method for genome-wide polyadenylation site mapping and RNA quantification.
abstract: The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.
authors.affiliation: Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wilkening
authors.forename: Vicent
authors.initials: V
authors.lastname: Pelechano
authors.forename: Aino I
authors.initials: AI
authors.lastname: Järvelin
authors.forename: Manu M
authors.initials: MM
authors.lastname: Tekkedil
authors.forename: Simon
authors.initials: S
authors.lastname: Anders
authors.forename: Vladimir
authors.initials: V
authors.lastname: Benes
authors.forename: Lars M
authors.initials: LM
authors.lastname: Steinmetz
mesh_terms: D002874:Chromosome Mapping; D020869:Gene Expression Profiling; D026723:Polyadenylation; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins