The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes
Eamens, A.L., Smith, N.A., Curtin, S.J., Wang, M.B., & Waterhouse, P.M. (2009) The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA, 15(12), pp. 2219-2235.
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 doublestranded 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 59 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.
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|Item Type:||Journal Article|
|Additional Information:||Articles free to read on journal website after 12 months|
|Keywords:||AGO1, DCL1, DCL4, DRB1, DRB4, Duplex strand selection, miRNA, RNA silencing, siRNA, Terminal thermostability, double stranded RNA, messenger RNA, microRNA, RNA binding protein, small interfering RNA, 5' untranslated region, Arabidopsis, article, biogenesis, bioinformatics, enzyme mechanism, gene silencing, mutant, Nicotiana benthamiana, nonhuman, Northern blotting, priority journal, protein cleavage, protein degradation, protein determination, protein expression, RNA sequence, RNA structure, thermostability, transgene, Arabidopsis Proteins, Base Sequence, Computational Biology, Gene Expression Regulation, Plant, MicroRNAs, RNA Stability, RNA, Double-Stranded, RNA-Binding Proteins, Tobacco, Arabidopsis thaliana, PMCID: PMC2779670|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
|Divisions:||Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Deposited On:||08 Jan 2014 03:35|
|Last Modified:||29 Jan 2015 05:53|
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