Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.     

Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates

Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ～70-85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.     

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

Dicer is a member of the ribonuclease III enzyme family and processes double‐stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non‐canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double‐stranded RNA‐binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA‐binding surface. The second dsRNA binding domain at C‐terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem‐loop structure of the RNA substrate, suggesting the possibility that stem‐loop RNA‐guided gene silencing pathway exists in budding yeast.     

Noncatalytic assembly of ribonuclease III with double-stranded RNA

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.     

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.     

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.     

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.     

The N-terminal domain that distinguishes yeast from bacterial RNase III contains a dimerization signal required for efficient double-stranded RNA cleavage

Yeast Rnt1 is a member of the double-stranded RNA (dsRNA)-specific RNase III family identified by conserved dsRNA binding (dsRBD) and nuclease domains. Comparative sequence analyses have revealed an additional N-terminal domain unique to the eukaryotic homologues of RNase III. The deletion of this domain from Rnt1 slowed growth and led to mild accumulation of unprocessed 25S pre-rRNA. In vitro, deletion of the N-terminal domain reduced the rate of RNA cleavage under physiological salt concentration. Size exclusion chromatography and cross-linking assays indicated that the N-terminal domain and the dsRBD self-interact to stabilize the Rnt1 homodimer. In addition, an interaction between the N-terminal domain and the dsRBD was identified by a two-hybrid assay. The results suggest that the eukaryotic N-terminal domain of Rnt1 ensures efficient dsRNA cleavage by mediating the assembly of optimum Rnt1-RNA ribonucleoprotein complex.     

Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants.

Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III.

The double-stranded RNA-specific endoribonuclease III (RNase III) of bacteria consists of an N-terminal nuclease domain and a double-stranded RNA binding domain (dsRBD) at the C-terminus. Analysis of two hybrid proteins consisting of the N-terminal half of Escherichia coli RNase III fused to the dsRBD of the Rhodobacter capsulatus enzyme and vice versa reveals that both domains in combination with the particular substrate determine substrate specificity and cleavage site selection. Extension of the spacer between the two domains of the E. coli enzyme from nine to 20 amino acids did not affect cleavage site selection.     

A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12–14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30–40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix α1, a key RNA-recognition element of the dsRBD.     

Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop

Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN. Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is reduced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD.     

Inorganic phosphate blocks binding of pre‐miRNA to Dicer‐2 via its PAZ domain

In Drosophila, Dicer‐1 produces microRNAs (miRNAs) from pre‐miRNAs, whereas Dicer‐2 generates small interfering RNAs from long double‐stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer‐2 cleavage of pre‐miRNAs, but not long dsRNAs. Here, we report that phosphate‐dependent substrate discrimination by Dicer‐2 reflects dsRNA substrate length. Efficient processing by Dicer‐2 of short dsRNA requires a 5′ terminal phosphate and a two‐nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer‐2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre‐miRNA processing in flies to Dicer‐1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme.     

A conserved major groove antideterminant for Saccharomyces cerevisiae RNase III recognition

Rnt1p, the only known Saccharomyces cerevisiae RNase III double-stranded RNA endonuclease, plays important roles in the processing of precursors of ribosomal RNAs and small nuclear and nucleolar RNAs and in the surveillance of unspliced pre-mRNAs. Specificity of cleavage by Rnt1p relies on the presence of RNA tetraloop structures with the consensus sequence AGNN at the top of the target dsRNA. The sequences of 79 fungal RNase III substrates were inspected to identify additional conserved sequence elements or antideterminants that may contribute to Rnt1p recognition of the double-stranded RNA. Surprisingly, U-A sequences at the base pair adjacent to the conserved terminal tetraloop (closing base pair) were found to be absent from all but one inspected sequence. Analysis of chemically modified variants of the closing base pair showed that the presence of exocyclic groups in the major groove of purines 3' to the last nucleotide of the tetraloop inhibits Rnt1p cleavage without strongly inhibiting Rnt1p binding. We propose that these groups interfere with the recognition of the RNA substrate by the catalytic domain of Rnt1p. These results identify exocyclic groups of purines in the major groove downstream of the tetraloop as a major antideterminant in S. cerevisiae RNase III activity, and suggest a rationale for their apparent counter selection in RNA processing sites.     

Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.     

Short RNA duplexes guide sequence-dependent cleavage by human Dicer

Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 3'-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 3'-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 2'-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic.     

The inside-out mechanism of Dicers from budding yeasts

The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function.     

Intermediate states of ribonuclease III in complex with double-stranded RNA

Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III*dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.