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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Main Authors:	Seong, Youngmo, Lim, Do-Hwan, Kim, Augustine, Seo, Jae Hong, Lee, Young Sik, Song, Hoseok, Kwon, Young-Soo
Format:	Online
Language:	English
Published:	Oxford University Press 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227787/

id	pubmed-4227787
recordtype	oai_dc
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 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
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo
spellingShingle	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
author_facet	Seong, Youngmo Lim, Do-Hwan Kim, Augustine Seo, Jae Hong Lee, Young Sik Song, Hoseok Kwon, Young-Soo
author_sort	Seong, Youngmo
title	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_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_sort	global identification of target recognition and cleavage by the microprocessor in human es cells
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.
publisher	Oxford University Press
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227787/
_version_	1613155073139933184

Global identification of target recognition and cleavage by the Microprocessor in human ES cells

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