Structural Insight into the Mechanism of Double-Stranded RNA Processing by Ribonuclease III

Abstract

Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.

Drosha and Dicer: Slicers cut from the same cloth

“…One small 23-aa DGCR8 helix (named as G1) interacts with each DROSHA RIIID, with the interaction between G1s and RIIIDs playing an important role in stabilizing DROSHA. The superposition of RNaseIII domains of Drosha with Aquifex aeolicus RNaseIII (AaRNaseIII) [11] indicates that DRO-SHA RIIIDs utilize a canonical catalytic mechanism involving two metal ions at the catalytic sites, thereby confirming previous predictions that a pair of RIIIDs generate a composite processing center. However, there is an unusual long conserved insertion (aa 898-964) positioned within the DROSHA RIIIDa predicted to involve two helical segments.…”

“…In order to illustrate how Microprocessor could potentially recognize substrate pri-miRNAs, Kwon et al built a model of the pri-miRNA bound to DROSHA-DGCR8 ( Figure 1D), with the model guided by the structure of dsRNA-bound AaRNase III [11]. Given the directionality of the DGCR8 CTT domain relative to the DROSHA RIIIDs, it is conceivable that full-length DGCR8 can form a symmetrically elongated complex, such that dimerized Rhed and dsRBD domains are positioned to interact with the upper stem and apical loop of bound pri-miRNAs.…”

Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila

“…However, normal Dcr-2 levels in the dcr-2 P1496L mutant suggest the importance of P1496 in Dcr-2 RNase III activity. The crystal structures of the Aquifex aeolicus (Aa) RNase III-product complex and the protozoan Giardia Dicer [11,12], along with the biochemical analysis of recombinant human Dicer [13], have provided a single processing-center model for Dicer. In this model, two RNase III domains of Dicer form an intramolecular pseudodimer that resembles the homodimer of bacterial RNase III enzymes [14], and create a catalytic valley in which dsRNA substrates are cleaved by two catalytic sites on the opposite strands to generate products with 2-nt 3′ overhangs.…”

“…Additionally, the recent crystal structure of the single RNase IIIb domain of human Dicer showed that the domains could self-associate to form a stable homodimer, mimicking the intramolecular dimerization between the two RNase III domains of Dicer [15]. P1496 affected in the dcr-2 P1496L mutant is equivalent to the E64 residue of Aa-RNase III that is involved in substrate recognition and scissile-bond selection within the RNA-binding motif RBM 3 located on both ends of the catalytic valley [11], which is conserved in other RNase III domains (Fig. 1C).…”

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

“…The structures of different dsRBDs have been determined uncovering a mixed α/β fold with a conserved αβββα topology in which the two α-helices are packed against the three-stranded anti-parallel β-sheet [30,31]. In addition, structures of dsRBDs have been determined in complex with dsRNA, predominantly with non-natural RNA duplexes [32][33][34][35][36][37][38], revealing the canonical mode of dsRNA recognition by dsRBDs. Molecular recognition is made via three regions of interaction: helix α1 and the loop between β1 and β2 contact dsRNA minor grooves at one turn of interval whereas the short loop between β3 and α2 together with the N-terminal part of helix α2 contact the dsRNA phosphate backbone across the major groove.…”