Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Welch, Joshua D., Slevin, Michael K., Tatomer, Deirdre C., Duronio, Robert J., Prins, Jan F., Marzluff, William F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2015
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
_version_ 1782377874134663168
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
work_keys_str_mv AT welchjoshuad endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates
AT slevinmichaelk endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates
AT tatomerdeirdrec endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates
AT duroniorobertj endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates
AT prinsjanf endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates
AT marzluffwilliamf endseqandappendsequencing3endstoidentifynontemplatedtailsanddegradationintermediates