The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

Andrew Eamens; Neil A. Smith; Shaun J. Curtin; Ming-Bo Wang; Peter M. Waterhouse

doi:10.1261/rna.1646909

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The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

Journal article

Peer reviewed

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

Andrew Eamens, Neil A. Smith, Shaun J. Curtin, Ming-Bo Wang and Peter M. Waterhouse

RNA, Vol.15(12), pp.2219-2235

2009

DOI: https://doi.org/10.1261/rna.1646909

PMCID: PMC2779670

PMID: 19861421

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Abstract

miRNA

siRNA

terminal thermostability

duplex strand selection

DRB1

DCL1

DRB4

DCL4

AGO1

RNA silencing

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5' end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 5' thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

Details

Title: The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes
Authors: Andrew Eamens (Author) - CSIRO Plant Industry
Neil A. Smith (Author) - CSIRO Plant Industry
Shaun J. Curtin (Author) - CSIRO Plant Industry
Ming-Bo Wang (Author) - CSIRO Plant Industry
Peter M. Waterhouse (Author) - CSIRO Plant Industry
Publication details: RNA, Vol.15(12), pp.2219-2235
Publisher: Cold Spring Harbor Laboratory Press
DOI: 10.1261/rna.1646909
ISSN: 1469-9001
PMID: 19861421; PMC2779670
Organisation Unit: School of Health and Behavioural Sciences - Legacy; University of the Sunshine Coast, Queensland; School of Health - Biomedicine
Language: English
Record Identifier: 99656589602621
Output Type: Journal article

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Collaboration types: Domestic collaboration
Web Of Science research areas: Biochemistry & Molecular Biology

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