Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The stability of mRNA in prokaryotes depends on multiple factors and it has not yet been possible to describe the process of mRNA degradation in terms of a unique pathway. However, important advances have been made in the past 10 years with the characterization of the cis-acting RNA elements and the trans-acting cellular proteins that control mRNA decay. The trans-acting proteins are mainly four nucleases, two endo- (RNase E and RNase III) and two exonucleases (PNPase and RNase II), and poly(A) polymerase. RNase E and PNPase are found in a multienzyme complex called the degradosome. In addition to the host nucleases, phage T4 encodes a specific endonuclease called RegB. The cis-acting elements that protect mRNA from degradation are stable stem-loops at the 5' end of the transcript and terminators or REP sequences at their 3' end. The rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage that often occurs at the 5' extremity. This initial step is followed by directional 3' to 5' degradation by the two exonucleases. Several examples, reviewed here, indicate that mRNA degradation is an important step at which gene expression can be controlled. This regulation can be either global, as in the case of growth rate-dependent control, or specific, in response to changes in the environmental conditions.
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PMID:Messenger RNA stability and its role in control of gene expression in bacteria and phages. 1069 Apr 8

RNase III endonucleases cleave double-stranded RNA, transforming precursor RNAs into mature RNAs that act in pre-mRNA splicing, RNA modification, translation, gene silencing, and the regulation of developmental timing. The recently solved structure of an RNase III endonuclease domain provides a hint at how this family of ribonucleases functions.
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PMID:Thirty-three years later, a glimpse at the ribonuclease III active site. 1188 96

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.
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PMID:Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. 1241 5

Rnt1p, the only known Saccharomyces cerevisiae RNase III endonuclease, plays important functions in the processing of precursors of rRNAs (pre-rRNAs) and of a large number of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). While most eukaryotic RNases III, including the Schizosaccharomyces pombe enzyme Pac1p, cleave double-stranded RNA without sequence specificity, Rnt1p cleavage relies on the presence of terminal tetraloop structures that carry the consensus sequence AGNN. To search for the conservation of these processing signals, I have systematically analyzed predicted secondary structures of the 3' external transcribed spacer (ETS) sequences of the pre-rRNAs and of flanking sequences of snRNAs and snoRNAs from sequences available in 13 other Hemiascomycetes species. In most of these species, except in Yarrowia lipolytica, double-stranded RNA regions capped by terminal AGNN tetraloops can be found in the 3' ETS sequences of rRNA, in the 5'- or 3'-end flanking sequences of sn(o)RNAs, or in the intergenic spacers of polycistronic snoRNA transcription units. This analysis shows that RNase III processing signals and RNase III cleavage specificity are conserved in most Hemiascomycetes species but probably not in the evolutionarily more distant species Y. lipolytica.
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PMID:Conservation of RNase III processing pathways and specificity in hemiascomycetes. 1455 72

Small nucleolar RNAs (snoRNAs) play important roles in ribosomal RNA metabolism. In Saccharomyces cerevisiae, box C/D snoRNAs are synthesized from excised introns, polycistronic precursors, or independent transcription units. Previous studies have shown that only a few independently transcribed box C/D snoRNAs are processed at their 5' end. Here we describe 12 additional independently transcribed box C/D snoRNAs that undergo 5'-end processing. 5' Extensions found in the precursors of these snoRNAs contain cleavage sites for Rnt1p, the S. cerevisiae homolog of RNase III, and unprocessed precursors accumulate in vivo in the absence of Rnt1p. Rnt1p cleavage products were identified in vivo when the 5' --> 3' exonucleases Xrn1p and Rat1p are inactivated (xrn1delta rat1-1) and in vitro using model RNA substrates and recombinant Rnt1p. Some of these snoRNAs show increased levels of unprocessed precursors when the rnt1Delta deletion is combined to the xrn1delta rat1-1 mutation, suggesting that these exonucleases participate in the 5' processing or the degradation of the snoRNA precursors. Unprocessed precursors are not significantly destabilized in the absence of the trimethylguanosine capping enzyme Tgs1p, suggesting that a 5' monomethyl cap is sufficient to ensure stabilization of these precursors. These results demonstrate that the majority of independently transcribed box C/D snoRNAs from the yeast genome undergo 5'-end processing and that the Rnt1p endonuclease and the Xrn1p and Rat1p 5' --> 3'exonucleases have partially redundant functions in the 5'-end processing of these snoRNAs.
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PMID:The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5'-end processing of S. cerevisiae box C/D snoRNAs. 1456 86

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.
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PMID:Noncatalytic assembly of ribonuclease III with double-stranded RNA. 1506 73

Specific recognition of double-stranded RNA (dsRNA) by dsRNA-binding domains (dsRBDs) is involved in a large number of biological and regulatory processes. Although structures of dsRBDs in complex with dsRNA have revealed how they can bind to dsRNA in general, these do not explain how a dsRBD can recognize specific RNAs. Rnt1p, a member of the RNase III family of dsRNA endonucleases, is a key component of the Saccharomyces cerevisiae RNA-processing machinery. The Rnt1p dsRBD has been implicated in targeting this endonuclease to its RNA substrates, by recognizing hairpins closed by AGNN tetraloops. We report the solution structure of Rnt1p dsRBD complexed to the 5' terminal hairpin of one of its small nucleolar RNA substrates, the snR47 precursor. The conserved AGNN tetraloop fold is retained in the protein-RNA complex. The dsRBD contacts the RNA at successive minor, major, and tetraloop minor grooves on one face of the helix. Surprisingly, neither the universally conserved G nor the highly conserved A are recognized by specific hydrogen bonds to the bases. Rather, the N-terminal helix fits snugly into the minor groove of the RNA tetraloop and top of the stem, interacting in a non-sequence-specific manner with the sugar-phosphate backbone and the two nonconserved tetraloop bases. Mutational analysis of residues that contact the tetraloop region show that they are functionally important for RNA processing in the context of the entire protein in vivo. These results show how a single dsRBD can convey specificity for particular RNA targets, by structure specific recognition of a conserved tetraloop fold.
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PMID:Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III. 1515 Apr 9

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.
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PMID:Single processing center models for human Dicer and bacterial RNase III. 1524 44

Eukaryotes have two types of ribosomes containing either 5.8SL or 5.8SS rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8SL rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8SS rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8SL rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Delta3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Delta3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8SS and 5.8SL rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed.
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PMID:The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA. 1552 10

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg2+ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg2+ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n (H)) is 2.0 +/- 0.1, indicative of the involvement of two Mg2+ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione--an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites--inhibits E.coli RNase III cleavage of R1.1 RNA. The IC50 for the compound is 14 microM for the Mg2+-supported reaction, and 8 microM for the Mn2+-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.
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PMID:Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis. 1569 82


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