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Review
. 2014 Jan-Feb;5(1):31-48.
doi: 10.1002/wrna.1195. Epub 2013 Sep 30.

Ribonuclease III mechanisms of double-stranded RNA cleavage

Allen W Nicholson  1
Affiliations
Free PMC article
Review

Ribonuclease III mechanisms of double-stranded RNA cleavage

Allen W Nicholson. Wiley Interdiscip Rev RNA. 2014 Jan-Feb.
Free PMC article

Abstract

Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the ribonuclease III (RNase III) family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3'-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a noncatalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA.

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Figures

Figure 1
Figure 1
Domain structures of ribonuclease III (RNase III) family polypeptides. Domain key: RIII (gray), RNase III domain; dsRBD (light blue), dsRNA-binding domain; PR (orange), proline-rich domain; RS (Blue), arginine/serine-rich domain; DExD/H (blue), helicase domain; DUF (brown), DUF283 domain; PF (green), platform domain; PAZ (purple), PAZ domain; C2H2 (pink): C2H2 Zinc finger domain; PUF (yellow), Pumilio/FBF homology domain. The blue double-headed arrow between the PAZ and RIIIa domains of human and giardia Dicer indicates the dsRNA ‘measuring’ segment. The parentheses in the PUF domain of mRPN1 indicate that the identity of the domain has not been confirmed. For RNC1, the red letters ‘X’ refer to inactivating point mutations of catalytic site residues. Diagrams are not to scale.
Figure 2
Figure 2
Ribonuclease III (RNase III) catalytic site structure. Shown are the interactions, presumably subsequent to phosphodiester hydrolysis, in the catalytic site of the 1.7 Å structure of wild-type Aa-RNase III bound to cleaved dsRNA (PDB entry 2NUG). The two Mg2+ ions (A and B) are shown in green and are coordinated to the E40, D44, D107, and E110 side chains. Metal-oxygen coordinate bonds are shown as dotted black lines. The oxygen atom of the water nucleophile that is bonded to phosphorus (yellow-gold) is colored in lavender. Water molecules are shown as red spheres. The nucleotides (R-1, R-0, and R+1) are colored in blue (nitrogen), black (carbon) and red (oxygen), and the numbering refers to their position with respect to the scissile phosphodiester (between R-0 and R-1).
Figure 3
Figure 3
Ribonuclease III (RNase III) structures. (a) 2.0 Å structure of Thermotoga maritima RNase III, determined by the Joint Center for Structural Genomics, University of California at San Diego (PDB entry 1O0w). The structure displays the two dsRBDs (dark orange) in symmetric, extended positions with respect to the RNase III domain (green). The flexible linkers are shown in yellow. (b) A 1.7 Å structure of Aquifex aeolicus (Aa) RNase III bound to dsRNA (PDB entry 2NUG). The color scheme in panel (a) also is used here, and the two Mg2+ ions in each catalytic site are colored in cyan. RNA is shown in gray. The phosphodiester bonds at each catalytic site have been hydrolyzed, so the complex contains two dsRNA segments.
Figure 4
Figure 4
Proposed catalytic pathway for ribonuclease III (RNase III). The diagram is a modification of a proposed scheme., Initial recognition of dsRNA (black) by a dsRBD (pink) is followed by engagement of the RNase III domain (light blue) and the second dsRBD to form a precatalytic complex. Phosphodiester hydrolysis (red arrows) provides a post-catalytic complex with products still bound by the RNase III domain. Release of the products from the RNase III domain is followed by release from the dsRBDs. The rate-limiting step may be release of products from the RNase III domain (see text). (Reprinted with permission from Ref . Copyright 2011 Springer Science+Business Media).
Figure 5
Figure 5
Reactivity determinants of ribonuclease III (RNase III) substrates. (a) Diagrammatic structure of an idealized dsRNA substrate of Ec-RNase III. The length of the helix from the end of each db is 22 bp (two A-helical turns). The boxed bp indicate the distal box (db), middle box (mb) and proximal box (pb). The twofold symmetry of the substrate is indicated by the black dot centered between the two cleavage sites (black arrows), and blue color of the lower portion of the substrate. Note that the symmetry does not include specific bp sequence. N-N′ indicate a standard bp of unspecified sequence. The sites of interaction of RBM1, RBM2, RBM3, and RBM4 with the db, mb, and pb are indicated by dashed lines. (b) Sequence-dependent interaction of the conserved glutamine side chain of the dsRBD α1 helix (RBM1) with the O2 uracil atom. Taken from the Aa-RNase III(D44N)-dsRNA crystal structure (PDB entry 2EZ6). The dotted lines indicate probable hydrogen bonds of the Q157 carboxamide group with the O2 atom and the adjacent 2′-hydroxyl group.
Figure 6
Figure 6
Reactivity determinants of a pri-miRNA substrate for Drosha, and a proposed DGCR8-dsRNA interaction. (a) Features of mammalian pri-miR-16-1 RNA important for reactivity. The grey lines indicate the positions of the miRNA sequences, and the Drosha cleavage sites are at the left hand termini of the lines, that upon hydrolysis create 2-nt, 3′-overhang product ends (see text). ‘ss/ds junction’ indicates the region recognized by DGCR8, and ‘Hot Spot’ refers to a deformable region proximal to the Drosha cut site. H1, H2, H3 denote regular helical regions that are punctuated by the deformable regions. The color scheme for individual nucleotides (solid circles) indicates the relative probability of single-strandedness, determined as described: blue, low probability; green/yellow, medium probability; red, high probability. (Reprinted with permission from Ref . Copyright 2013 American Chemical Society). (b) Proposed model of a complex of dsRNA bound to the DGCR8 core, consisting of dsRBD1, dsRBD2, the connecting linker, and the C-terminal helix. The fixed relative positions of the two dsRBDs and the corresponding placement of the respective dsRNA-binding surfaces suggest that engagement of a pri-miRNA by the DGCR8 polypeptide requires distortion of the pri-miRNA structure, which would be facilitated by the deformable regions (see panel (a))., (Reprinted with permission from Ref . Copyright 2004 Macmillan Publishers Ltd)
Figure 7
Figure 7
Structural features of a ‘minimal’ Dicer. (a) Two views of the 3.3 Å crystal structure of Dicer from Giardia intestinalis (PDB entry 2ffl). The PAZ domain (gold) is linked to the RNase IIIa domain (yellow) by the connector helix, or ‘ruler’ (red) (see also Figure 1), which determines the dsRNA product length. The RNase IIIb domain is shown in green, and the two catalytic sites (within the rectangle) are identified by the two metal (Er3+) ions (purple) in each site (see also Figure 2). (b) Two views of a modeled complex of gDicer bound to dsRNA. Blue and red coloration indicate basic and acidic regions, respectively. dsRNA is shown in yellow and gray strands, and the white arrows indicate the sites of cleavage. The yellow star indicates the site in the PAZ domain that binds the dsRNA 3′-overhang. (Reprinted with permission from Ref . Copyright 2006 AAAS)
Figure 8
Figure 8
Functional interplay of human(h) Dicer domains in dsRNA processing. (a) Domain structure of hDicer as determined by domain-specific tagging and single molecule electron microscopy (EM). Shown are the PAZ (purple), platform (blue), ruler (gray), dsRBD (blue), ribonuclease III (RNase III) (a+b) (orange) and helicase domains, with the latter domain displayed as three subdomains HEL1 (red), HEL2 (blue), and HEL2i (orange). (b) Two views of hDicer bound to a pre-miRNA hairpin, showing the engagement of the loop by the helicase domain, and the opposing 3′-overhang end by the PAZ domain. (c) Proposed processing cycle for hDicer cleavage of dsRNA. In complex 1, the dsRNA is engaged by the helicase domain. In complex 2, cooperation of the PAZ and RNase III domains provide a 22 bp product, while the remainder of the dsRNA remains engaged with the helicase domain. In complex 3, release of the 22 bp product from the PAZ and RNase III domains allows subsequent engagement of the contiguous dsRNA segment by the RNase III and PAZ domains. This cycle explains the processivity of hDicer action, supported by the helicase domain. (Reprinted with permission from Ref . Copyright 2012 Macmillan Publishers Ltd)
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References

    1. Haines DS, Strauss KI, Gillespie DH. Cellular response to double-stranded RNA. J Cell Biochem. 1991;46:9–20. - PubMed
    1. Nicholson AW. Structure, reactivity and biology of double-stranded RNA. Prog Nucleic Acid Res Mol Biol. 1996;52:1–65. - PubMed
    1. Wang Q, Carmichael GG. Effects of length and location on the cellular response to double-stranded RNA. Microbiol Mol Biol Rev. 2004;68:432–452. - PMC - PubMed
    1. Gantier MP, Williams BR. The response of mammalian cells to double-stranded RNA. Cytokine Growth Factor Rev. 2007;18:363–371. - PMC - PubMed
    1. deFaria IJ, Olmo RP, Silva EG, Marques JT. dsRNA sensing during viral infection: lessons from plants, worms, insects, and mammals. J. Interferon Cytokine Res. 2013;33:239–253. - PubMed

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