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.     

Sequence dependence of substrate recognition and cleavage by yeast RNase III

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.     

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.     

Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III

RNase III enzymes form a conserved family of proteins that specifically cleave double-stranded (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. Yeast RNase III (Rnt1p) selects its substrate by recognizing the structure generated by a conserved NGNN tetraloop (G2-loop). Mutations of the invariant guanosine stringently inhibit binding and cleavage of all known Rnt1p substrates. Surprisingly, we have found that the 5' end of small nucleolar RNA 48 is processed by Rnt1p in the absence of a G2-loop. Instead, biochemical and structural analyses revealed that cleavage, in this case, is directed by a hairpin capped with an AAGU tetraloop, with a preferred adenosine in the first position (A1-loop). Chemical probing indicated that A1-loops adopt a distinct structure that varies at the 3' end where Rnt1p interacts with G2-loops. Consistently, chemical footprinting and chemical interference assays indicate that Rnt1p binds to G2 and A1-loops using different sets of nucleotides. Also, cleavage and binding assays showed that the N-terminal domain of Rnt1p aids selection of A1-capped hairpins. Together, the results suggest that Rnt1p recognizes at least two distinct classes of tetraloops using flexible protein RNA interactions. This underscores the capacity of double-stranded RNA binding proteins to use several recognition motifs for substrate identification.     

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.     

Mapping of possible binding sequences of two beta-sheet breaker peptides on beta amyloid peptide of Alzheimer's disease

Aggregation of amyloid peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease. Increased risk for disease is associated with increased formation of polymerized Abeta. Inhibition of formation of toxic (aggregated) form of Abeta is one of the therapeutic possibilities. Beta sheet breaker peptides (BSBs) fulfill the requirements of an effective inhibitor. After having attached to the Abeta molecules, BSBs can prevent aggregation of Abeta to polymeric forms (aggregates). In the present study, we performed molecular modelling of complex formation between Abeta and two BSB peptides. Our aim was to find proper binding sequences for the BSB peptides on Abeta and characterize them. A dimeric model of Abeta was also used to study the interaction of BSBs with the aggregated forms of Abeta and find the sequences responsible for the polymerization process. A fast and efficient computational method: molecular docking was used for the afore-mentioned purposes.     

Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease.

An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre-mRNAs, flanks the majority of yeast intron-encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron-encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA- coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing-deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis, but does not affect processing of a dicistronic snoRNA-coding unit that has a canonical Rnt1p site. Correct cleavage of intron-encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull-down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron-encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred.     

Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs

dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.     

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.     

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.     

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.     

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.