Solution structure of the Drosha double-stranded RNA-binding domain

Mueller, Geoffrey A.; Miller, Matthew T.; DeRose, Eugene F.; Ghosh, Mahua; London, Robert E.; Hall, Traci M. Tanaka

doi:10.1186/1758-907x-1-2

Cited by 24 publications

(24 citation statements)

References 37 publications

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“…We next aimed to confirm that the backbone dynamics of these two constructs are also consistent through measurement of 15 N-spin relaxation (Fig 3). Reassuringly, qualitative trends in the calculated order parameters for wt-Drosha-dsRBD and Drosha-Quad are nearly identical (Fig 3A) and both are consistent with estimates of ps-ns dynamics established by [ 1 H], 15 N-NOE values independently reported for Drosha-dsRBD [19]. While there is a slight baseline offset in the order parameter profiles, this may be attributable to subtle changes in the diffusion tensor for Drosha-Quad and therefore is not deemed large enough to be significant.…”

Section: Resultssupporting

confidence: 88%

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

et al. 2017

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Canonical processing of miRNA begins in the nucleus with the Microprocessor complex, which is minimally composed of the RNase III enzyme Drosha and two copies of its cofactor protein DGCR8. In structural analogy to most RNase III enzymes, Drosha possesses a modular domain with the double-stranded RNA binding domain (dsRBD) fold. Unlike the dsRBDs found in most members of the RNase III family, the Drosha-dsRBD does not display double-stranded RNA binding activity; perhaps related to this, the Drosha-dsRBD amino acid sequence does not conform well to the canonical patterns expected for a dsRBD. In this article, we investigate the impact on miRNA processing of engineering double-stranded RNA binding activity into Drosha’s non-canonical dsRBD. Our findings corroborate previous studies that have demonstrated the Drosha-dsRBD is necessary for miRNA processing and suggest that the amino acid composition in the second α-helix of the domain is critical to support its evolved function.

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Section: Resultssupporting

confidence: 88%

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

et al. 2017

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“…The portion of the protein N-terminal to its paired catalytic domains is typical of a Class 2 RNase III in that it contains a proline-rich region, but the function of this portion remains unknown (Figure 2 a ). Drosha’s C-terminal dsRBD is canonical in its sequence and tertiary structure (59) and is required for pri-miRNA processing in vivo (2) but does not appear to play a substantial role in substrate binding or recognition in vitro (95), tasks instead performed by DGCR8 (30). Accordingly, Drosha cleaves pri-miRNAs indiscriminately in the absence of DGCR8 (26).…”

Section: Activity Structure and Interactions Of Mirna Pathway Proteinsmentioning

confidence: 99%

Molecular Mechanisms of RNA Interference

Wilson

Doudna

2013

Annu. Rev. Biophys.

919

694

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Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral infection. Specialized ribonucleases and RNA binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha, pre-miRNAs are transported to the cytoplasm, where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins, and RNA-bound complexes, have offered exciting insight into the mechanisms operating at the heart of RNA silencing pathways.

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“…31 Additionally, some dsRBDs have a canonical dsRBD fold but do not independently bind to dsRNA with high affinity, such as the human Drosha dsRBD. 32 …”

Section: Introductionmentioning

confidence: 99%

Intrinsic Dynamics of an Extended Hydrophobic Core in the S. cerevisiae RNase III dsRBD Contributes to Recognition of Specific RNA Binding Sites

Hartman

Wang

Zhang

et al. 2013

Journal of Molecular Biology

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The S. cerevisiae RNase III enzyme Rnt1p preferentially binds to dsRNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its dsRBD helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin relaxation experiments. The intrinsic μs-ms timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1-β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and non-specific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity, and cause defects in snoRNA processing in vivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.

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Solution structure of the Drosha double-stranded RNA-binding domain

Cited by 24 publications

References 37 publications

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

Molecular Mechanisms of RNA Interference

Intrinsic Dynamics of an Extended Hydrophobic Core in the S. cerevisiae RNase III dsRBD Contributes to Recognition of Specific RNA Binding Sites

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