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Global identification of target recognition and cleavage by the Microprocessor in human ES cells
The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addresse...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227787/ https://www.ncbi.nlm.nih.gov/pubmed/25326327 http://dx.doi.org/10.1093/nar/gku957 |
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author | Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo |
author_facet | Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo |
author_sort | Seong, Youngmo |
collection | PubMed |
description | The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. |
format | Online Article Text |
id | pubmed-4227787 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-42277872014-11-21 Global identification of target recognition and cleavage by the Microprocessor in human ES cells Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo Nucleic Acids Res RNA The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. Oxford University Press 2014-11-10 2014-10-17 /pmc/articles/PMC4227787/ /pubmed/25326327 http://dx.doi.org/10.1093/nar/gku957 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | RNA Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title | Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title_full | Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title_fullStr | Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title_full_unstemmed | Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title_short | Global identification of target recognition and cleavage by the Microprocessor in human ES cells |
title_sort | global identification of target recognition and cleavage by the microprocessor in human es cells |
topic | RNA |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227787/ https://www.ncbi.nlm.nih.gov/pubmed/25326327 http://dx.doi.org/10.1093/nar/gku957 |
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