Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis

Rikako Hirata; Tomoya Makabe; Kei ichiro Mishiba; Nozomu Koizumi; Samir M. Hamdan; Yuji Iwata

doi:10.1111/gtc.12927

Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis

Rikako Hirata, Tomoya Makabe, Kei ichiro Mishiba, Nozomu Koizumi, Samir M. Hamdan, Yuji Iwata^*

^*Corresponding author for this work

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

2 Scopus citations

Abstract

Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

Original language	English (US)
Pages (from-to)	280-292
Number of pages	13
Journal	Genes to Cells
Volume	27
Issue number	4
DOIs	https://doi.org/10.1111/gtc.12927
State	Published - Apr 2022

Bibliographical note

Funding Information:
This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H.

Funding Information:
This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H. The authors thank The Arabidopsis Information Resource (TAIR) for the gene annotation data. We thank Ayumi Sakei (Osaka Prefecture University) for technical assistance.

Publisher Copyright:
© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Keywords

Arabidopsis thaliana
Dicer
microRNA
microRNA biogenesis
RNA secondary structure

ASJC Scopus subject areas

Genetics
Cell Biology

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10.1111/gtc.12927

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@article{d6c8979ce80f4617bceadce4b1ceffd9,

title = "Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis",

abstract = "Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.",

keywords = "Arabidopsis thaliana, Dicer, microRNA, microRNA biogenesis, RNA secondary structure",

author = "Rikako Hirata and Tomoya Makabe and Mishiba, {Kei ichiro} and Nozomu Koizumi and Hamdan, {Samir M.} and Yuji Iwata",

note = "Funding Information: This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H. Funding Information: This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H. The authors thank The Arabidopsis Information Resource (TAIR) for the gene annotation data. We thank Ayumi Sakei (Osaka Prefecture University) for technical assistance. Publisher Copyright: {\textcopyright} 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.",

year = "2022",

month = apr,

doi = "10.1111/gtc.12927",

language = "English (US)",

volume = "27",

pages = "280--292",

journal = "Genes to Cells",

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AU - Hirata, Rikako

AU - Makabe, Tomoya

AU - Mishiba, Kei ichiro

AU - Koizumi, Nozomu

AU - Hamdan, Samir M.

AU - Iwata, Yuji

N1 - Funding Information: This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H. Funding Information: This work was supported by JSPS KAKENHI Grand Numbers JP17K15416 to Y.I. and JP20J15594 to R.H. The authors thank The Arabidopsis Information Resource (TAIR) for the gene annotation data. We thank Ayumi Sakei (Osaka Prefecture University) for technical assistance. Publisher Copyright: © 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

PY - 2022/4

Y1 - 2022/4

N2 - Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

AB - Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

KW - Arabidopsis thaliana

KW - Dicer

KW - microRNA

KW - microRNA biogenesis

KW - RNA secondary structure

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M3 - Article

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AN - SCOPUS:85124905468

SN - 1356-9597

VL - 27

SP - 280

EP - 292

JO - Genes to Cells

JF - Genes to Cells

IS - 4

ER -

Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis

Abstract

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Keywords

ASJC Scopus subject areas

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