Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

S. Chul Kwon; S. Chan Baek; Yeon Gil Choi; Jihye Yang; Young suk Lee; Jae Sung Woo; V. Narry Kim

doi:10.1016/j.molcel.2018.11.005

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

S. Chul Kwon, S. Chan Baek, Yeon Gil Choi, Jihye Yang, Young suk Lee, Jae Sung Woo, V. Narry Kim

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

55 Citations (Scopus)

Abstract

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.

Original language	English
Pages (from-to)	505-518.e5
Journal	Molecular Cell
Volume	73
Issue number	3
DOIs	https://doi.org/10.1016/j.molcel.2018.11.005
Publication status	Published - 2019 Feb 7

Bibliographical note

Funding Information:
We thank Hyeshik Chang and Yoonseok Jung for the bioinformatical supervision, Haedong Kim and Jimi Kim for sharing the optimized small RNA sequencing protocol, Kijun Kim for sharing unpublished results, Baekgyu Kim for sharing the HA-tagged DROSHA plasmid, and Seungmi Ji, Daeun Choi, Eunji Kim, and Young Yoon Lee for technical help. This research was supported by IBS-R008-D1 of the Institute for Basic Science funded by the Ministry of Science and ICT .

Publisher Copyright:
© 2018 Elsevier Inc.

Keywords

DGCR8
DROSHA
RNase III
miRNA
microRNA

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1016/j.molcel.2018.11.005

Cite this

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title = "Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing",

abstract = "Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.",

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author = "Kwon, {S. Chul} and Baek, {S. Chan} and Choi, {Yeon Gil} and Jihye Yang and Lee, {Young suk} and Woo, {Jae Sung} and Kim, {V. Narry}",

note = "Funding Information: We thank Hyeshik Chang and Yoonseok Jung for the bioinformatical supervision, Haedong Kim and Jimi Kim for sharing the optimized small RNA sequencing protocol, Kijun Kim for sharing unpublished results, Baekgyu Kim for sharing the HA-tagged DROSHA plasmid, and Seungmi Ji, Daeun Choi, Eunji Kim, and Young Yoon Lee for technical help. This research was supported by IBS-R008-D1 of the Institute for Basic Science funded by the Ministry of Science and ICT . Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",

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AU - Yang, Jihye

AU - Lee, Young suk

AU - Woo, Jae Sung

AU - Kim, V. Narry

N1 - Funding Information: We thank Hyeshik Chang and Yoonseok Jung for the bioinformatical supervision, Haedong Kim and Jimi Kim for sharing the optimized small RNA sequencing protocol, Kijun Kim for sharing unpublished results, Baekgyu Kim for sharing the HA-tagged DROSHA plasmid, and Seungmi Ji, Daeun Choi, Eunji Kim, and Young Yoon Lee for technical help. This research was supported by IBS-R008-D1 of the Institute for Basic Science funded by the Ministry of Science and ICT . Publisher Copyright: © 2018 Elsevier Inc.

PY - 2019/2/7

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N2 - Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.

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Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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