Open AccessAn efficient method for genome-wide polyadenylation site mapping and RNA quantification
Author(s) -
Stefan Wilkening,
Vicent Pelechano,
Aino I Järvelin,
Manu M. Tekkedil,
Simon Anders,
Vladimír Benes̆,
Lars M. Steinmetz
Publication year - 2013
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gks1249
Subject(s) - polyadenylation , biology , untranslated region , computational biology , saccharomyces cerevisiae , three prime untranslated region , rna , genetics , coding region , genome , translation (biology) , messenger rna , gene
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.