Second Double-Stranded RNA Binding Domain of Dicer-like Ribonuclease 1: Structural and Biochemical Characterization

Burdisso, Paula; Suarez, Irina Paula; Bologna, Nicolas Gerardo; Palatnik, Javier F.; Bersch, Beate; Rasia, Rodolfo M.

doi:10.1021/bi301247r

Cited by 19 publications

(17 citation statements)

References 43 publications

(93 reference statements)

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“…We then determined the binding affinities of both constructs for substrate RNA by means of fluorescence polarization assays (Figure 1 and Table 1 ). We tested two different regions of the miR172a precursor as binding partners, the lower stem region (pri-miR172a-ls), that was previously shown to be essential for the correct processing of miRNA precursors ( 25 ), and the miRNA/miRNA* region (miR172a stem-loop).The affinity of the isolated DCL1-A construct for pri-miR172a fragments is similar to the one determined previously for DCL1-B ( 13 ). DCL1-A RNA binding affinity is somewhat higher for the pri-miR172a-ls construct than for the miR172a stem loop.…”

Section: Resultsmentioning

confidence: 74%

“…More recently, a construct containing the two tandem dsRBDs of DCL1 was shown to complement hyl1–2 mutant plants, further demonstrating the importance of this region in substrate recognition and in the formation of the miRNA processing complex in plants ( 12 ). In a previous work we characterized the second dsRBD of DCL1 from Arabidopsis thaliana (DCL1-B hereafter) ( 13 ). Here we focus on the functional and structural features of the first dsRBD of the same protein.…”

Section: Introductionmentioning

confidence: 99%

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Induced folding in RNA recognition byArabidopsis thalianaDCL1

et al. 2015

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DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.

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Section: Resultsmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Induced folding in RNA recognition byArabidopsis thalianaDCL1

et al. 2015

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“…complement hyl1 phenotypes. By contrast, DCL1-D2 is not a canonical dsRBD, which may leave nucleic acid binding as a vestigial feature during evolution (Burdisso et al, 2012). DCL1-D2 may bind weakly with pri-miRNAs at a sensitivity below R-EMSA (Fig.…”

Section: Differential Functions Of Dsrbds Of Dcl1mentioning

confidence: 99%

Complementation of Hyponastic Leaves1 by Double-Strand RNA-Binding Domains of Dicer-Like1 in Nuclear Dicing Bodies

et al. 2013

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ORCID ID: 0000-0002-7391-0246 (Y.F.).MicroRNAs (miRNAs) are a class of small regulatory RNAs that are found in almost all of the eukaryotes. Arabidopsis (Arabidopsis thaliana) miRNAs are processed from primary miRNAs (pri-miRNAs), mainly by the ribonuclease III-like enzyme DICER-LIKE1 (DCL1) and its specific partner, HYPONASTIC LEAVES1 (HYL1), a double-strand RNA-binding protein, both of which contain two doublestrand RNA-binding domains (dsRBDs). These dsRBDs are essential for miRNA processing, but the functions of them are not clear. Here, we report that the two dsRBDs of DCL1 (DCL1-D1D2), and to some extent the second dsRBD (DCL1-D2), complement the hyl1 mutant, but not the first dsRBD of DCL1 (DCL1-D1). DCL1-D1 is diffusely distributed throughout the nucleoplasm, whereas DCL1-D2 and DCL1-D1D2 concentrate in nuclear dicing bodies in which DCL1 and HYL1 colocalize. We show further that protein-protein interaction is mainly mediated by DCL1-D2, while DCL1-D1 plays a major role in binding of pri-miRNAs. These results suggest parallel roles between C-terminal dsRBDs of DCL1 and N-terminal dsRBDs of HYL1 and support a model in which Arabidopsis pri-miRNAs are recruited to dicing bodies through functionally divergent dsRBDs of microprocessor for accurate processing of plant pri-miRNAs.

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“…In this way we can rationalize the similar impact on both function and affinity of both mutations introduced. It was suggested that electrostatic interactions play an important role in dsRBD-RNA recognition [37] , [40] although these effects seem to be dissimilar between different dsRBDs [41] . The absence of alterations in stability on the mutant proteins that could hinder RNA recognition highlight that phosphate backbone recognition is essential for RNA binding by these domains.…”

Section: Discussionmentioning

confidence: 99%

Structural Determinants of Arabidopsis thaliana Hyponastic Leaves 1 Function In Vivo

et al. 2014

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MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.

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Second Double-Stranded RNA Binding Domain of Dicer-like Ribonuclease 1: Structural and Biochemical Characterization

Cited by 19 publications

References 43 publications

Induced folding in RNA recognition byArabidopsis thalianaDCL1

Induced folding in RNA recognition byArabidopsis thalianaDCL1

Complementation of Hyponastic Leaves1 by Double-Strand RNA-Binding Domains of Dicer-Like1 in Nuclear Dicing Bodies

Structural Determinants of Arabidopsis thaliana Hyponastic Leaves 1 Function In Vivo

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