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RNA. 2015 Jul;21(7):1375-89. doi: 10.1261/rna.048785.114. Epub 2015 May 26.

EnD-Seq and AppEnD: sequencing 3' ends to identify nontemplated tails and degradation intermediates.

Author information

1
Department of Computer Science, Curriculum in Bioinformatics and Computational Biology.
2
Department of Biochemistry and Biophysics.
3
Department of Biology.
4
Department of Biology, Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
5
Department of Computer Science, Curriculum in Bioinformatics and Computational Biology, marzluff@med.unc.edu prins@cs.unc.edu.
6
Curriculum in Bioinformatics and Computational Biology, Department of Biochemistry and Biophysics, Department of Biology, Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA marzluff@med.unc.edu prins@cs.unc.edu.

Abstract

Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3' ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3' ends contain post-transcriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3' end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3' ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nt at the 3' end of mature histone mRNA maintaining the length of the histone transcripts. Histone mRNAs in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3' additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.

KEYWORDS:

bioinformatics; high-throughput sequencing; histone mRNA; mRNA

PMID:
26015596
PMCID:
PMC4478355
DOI:
10.1261/rna.048785.114
[Indexed for MEDLINE]
Free PMC Article

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