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EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates
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 modifica...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478355/ https://www.ncbi.nlm.nih.gov/pubmed/26015596 http://dx.doi.org/10.1261/rna.048785.114 |
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author | Welch, Joshua D. Slevin, Michael K. Tatomer, Deirdre C. Duronio, Robert J. Prins, Jan F. Marzluff, William F. |
author_facet | Welch, Joshua D. Slevin, Michael K. Tatomer, Deirdre C. Duronio, Robert J. Prins, Jan F. Marzluff, William F. |
author_sort | Welch, Joshua D. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4478355 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-44783552016-07-01 EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates Welch, Joshua D. Slevin, Michael K. Tatomer, Deirdre C. Duronio, Robert J. Prins, Jan F. Marzluff, William F. RNA Methods 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. Cold Spring Harbor Laboratory Press 2015-07 /pmc/articles/PMC4478355/ /pubmed/26015596 http://dx.doi.org/10.1261/rna.048785.114 Text en © 2015 Welch et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Methods Welch, Joshua D. Slevin, Michael K. Tatomer, Deirdre C. Duronio, Robert J. Prins, Jan F. Marzluff, William F. EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title | EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title_full | EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title_fullStr | EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title_full_unstemmed | EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title_short | EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
title_sort | end-seq and append: sequencing 3′ ends to identify nontemplated tails and degradation intermediates |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478355/ https://www.ncbi.nlm.nih.gov/pubmed/26015596 http://dx.doi.org/10.1261/rna.048785.114 |
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