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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...

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Autores principales: Seong, Youngmo, Lim, Do-Hwan, Kim, Augustine, Seo, Jae Hong, Lee, Young Sik, Song, Hoseok, Kwon, Young-Soo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
Materias:
RNA
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.
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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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