RNase III is a double-stranded-RNA (dsRNA)-specific endoribonuclease that introduces staggered cuts on each side of the RNA helix (28). In bacteria, RNase III is involved in processing pre-rRNA, tRNA, and phage polycistronic mRNA (7). Depletion of RNase III perturbs the expression level of about 10% of the bacterial proteins, suggesting a global role in gene regulation (10). Two eukaryotic homologues of RNase III were experimentally identified in Saccharomyces cerevisiae (Rnt1) (2) and Schizosaccharomyces pombe (Pac1) (14,35,41). In addition, database searches revealed homologues in the worm, mouse, and human (5, 35). Rnt1 was shown both in vivo and in vitro to process pre-rRNA (2, 18), three small nuclear RNAs (snRNAs) (1, 4, 40), and several small nucleolar RNAs (snoRNAs) (5, 6, 31). Similarly, Pac1 cleaves the 3Ј end of U2 snRNA and the 3Ј end of 25S rRNA (36,37,43). Also, it has been suggested that Pac1 plays a role in cell division, mating, and sporulation (14, 41). RNase III, Rnt1, and Pac1 cleave duplex RNAs longer than 20 nucleotides in vitro while their primary targets in vivo are intramolecular stem-loop structures (2, 33, 37). The basic features of the RNA cleavage mechanism appear to be similar for all three ribonucleases, but differences also exist that prevent free substrate exchange and genetic complementation (37).Bacterial RNase III has two functionally and structurally separable subdomains: a C-terminal dsRNA-binding domain (dsRBD) and an N-terminal nuclease domain (8,17). The dsRBD motif is located in the last 74 amino acids (aa) and adopts a tertiary fold consisting of two helices separated by three ␤-strands (17). This tertiary structure is conserved throughout the family of dsRNA binding proteins including the RNA-dependent kinase (PKR) (27) and the Drosophila staufen protein (3). The isolated dsRBD from Escherichia coli RNase III binds RNA to form a RNA-protein complex (17; A. Nicholson, personal communication). The solution structure of the bacterial RNase III dsRBD (17) and the protein-RNA cocrystal structure of frog dsRNA binding protein A (38) suggest multiple RNA-protein contacts involving the two ␣-helices and the loop between the first two ␤-strands of the dsRBD. The structure of the N-terminal nuclease domain of RNase III is not known, but many mutations have helped identify the main features required for RNA cleavage (8, 28). The nuclease domain contains two stretches of conserved acidic amino acid residues at positions 37 to 47 and positions 60 to 74 (7, 28). These amino acids play either a key role in catalysis or an essential structural role. Mutations in these two regions abolish RNA cleavage without affecting RNA binding (21,28).Yeast Rnt1 shares with bacterial RNase III the main structural features of the nuclease domain and dsRBD, suggesting that they have similar functions (2). However, unlike the bacterial enzyme, eukaryotic Rnt1 possesses an N-terminal domain. The N-terminal domain constitutes 36% of the total Rnt1 protein with no significant homology to other eukaryotic...

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Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.The RNase III family consists of a growing number of enzymes that includes at least 33 bacterial and 22 eukaryotic enzymes (1-3). The members of this family regulate gene expression by processing and degrading cellular RNAs (1, 2, 4 -6). Based on protein structure, the RNase III family has been divided into 4 sub-classes (1). Class I includes all bacterial enzymes that possess both the classical nuclease domain and a dsRNA 1 binding domain (dsRBD). The class II enzymes are distinguished by the presence of an N-terminal extension and includes fungal RNase IIIs such as Saccharomyces cerevisiae Rnt1p (7) and Schizosaccharomyces pombe PacI (8). The class III enzymes possess two nuclease domains and include both plant and vertebrate enzymes. Class IV contains all Dicer-like enzymes involved in RNA-mediated interference and are distinguished by the presence of both N-terminal helicase and PAZ domains (6, 9). The homology between the different RNase IIIs varies between 20 to 84% depending on their evolutionary distance, suggesting a low level of primary structure conservation (1). This relatively low degree of conservation probably reflects the species specificity of RNase III, which prevents genetic complementation between members of the RNase III family (10).Despite the high selectivity of RNase III cleavage in vivo, many of the enzymes have been shown to cleave RNA duplexes with low sequence complexity in vitro (11)(12)(13)(...

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Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A-A or A-C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5' side of the loop, exposing their Watson-Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage.