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Nucleic Acids Res. 2013 Mar 1;41(5):e65. doi: 10.1093/nar/gks1249. Epub 2013 Jan 7.

An efficient method for genome-wide polyadenylation site mapping and RNA quantification.

Author information

  • 1Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

Erratum in

  • Nucleic Acids Res. 2013 Jul;41(12):6370.

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.

PMID:
23295673
[PubMed - indexed for MEDLINE]
PMCID:
PMC3597643
Free PMC Article
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