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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.

Background: MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly., Methodology/principal Findings: We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding., Conclusion/significance: We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded - double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.