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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.
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PMID:Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease. 761 61

Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.
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PMID:The role of endonucleases in the expression of ribonuclease II in Escherichia coli. 764 46

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.
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PMID:Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. 769 92

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.
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PMID:The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli. 805 23

The degradation process of the rpsO mRNA is one of the best characterised in E coli. Two independent degradation pathways have been identified. The first one is initiated by an RNase E endonucleolytic cleavage which allows access to the transcript by polynucleotide phosphorylase and RNase II. Cleavage by RNase E gives rise to an rpsO message lacking the stabilising hairpin of the primary transcript; this truncated mRNA is then degraded exonucleolytically from its 3' terminus. This pathway might be coupled to the translation of the message. The second pathway allows degradation of polyadenylated rpsO mRNA independently of RNase II, PNPase and RNase E. The ribonucleases responsible for degradation of poly(A) mRNAs under these conditions are not known. Poly(A) tails have been proposed to facilitate the degradation of structured RNA by polynucleotide phosphorylase. In contrast, we believe that removal of poly(A) by RNase II stabilises the rpsO mRNA harbouring a 3' hairpin. In addition to these two pathways, we have identified endonucleolytic cleavages which occur only in strains deficient for both RNase E and RNase III suggesting that these two endonucleases protect the 5' leader of the mRNA from the attack of unidentified ribonuclease(s). Looping of the rpsO mRNA might explain how RNase E bound at the 5' end can cleave at a site located just upstream the hairpin of the transcription terminator.
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PMID:Multiple degradation pathways of the rpsO mRNA of Escherichia coli. RNase E interacts with the 5' and 3' extremities of the primary transcript. 891 31

The Pac1 ribonuclease of Schizosaccharomyces pombe is a member of the RNase III family of double-strand-specific ribonucleases. To examine RNA structural features required for efficient cleavage by the Pac1 RNase, we tested a variety of double-stranded and hairpin RNAs as substrates for the enzyme. The Pac1 RNase required substrates that have a minimal helix length of about 20 base pairs. The enzyme cut both strands of the helix at sites separated by two base pairs. However, Pac1 was also able to make a single-stranded cleavage within an internal bulge of an authentic Escherichia coli substrate at the same site chosen by RNase III. Pac1 efficiently degraded the structurally complex adenovirus VA RNA(I), but was inactive against the short HIV-1 TAR RNA hairpin. These results indicate that the Pac1 RNase prefers straight, perfect helices, but it can tolerate internal bulges that do not distort the helix severely. Like its homologue from Saccharomyces cerevisiae, the Pac1 RNase cleaved at two in vivo RNA processing sites in a hairpin structure in the 3' external transcribed spacer of the S. pombe pre-rRNA, suggesting a role for the enzyme in rRNA maturation.
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PMID:Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe. 932 93

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.
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PMID:Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E. 991 89

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.
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PMID:RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA. 1032 33

The maturation and degradation of RNA molecules are essential features of the mechanism of gene expression, and provide the two main points for post-transcriptional regulation. Cells employ a functionally diverse array of nucleases to carry out RNA maturation and turnover. Viruses also employ cellular ribonucleases, or even use their own in their reproductive cycles. Studies on bacterial ribonucleases, and in particular those from Escherichia coli, are providing insight into ribonuclease structure, mechanism, and regulation. Ongoing biochemical and genetic analyses are revealing that many ribonucleases are phylogenetically conserved, and exhibit overlapping functional roles and perhaps common catalytic mechanisms. This article reviews the salient features of bacterial ribonucleases, with a focus on those of E. coli, and in particular, ribonuclease III. RNase III participates in a number of RNA maturation and RNA decay pathways, and is regulated by phosphorylation in the T7 phage-infected cell. Plasmid and phage RNAs, in addition to cellular transcripts, are RNase III targets. RNase III orthologues occur in eukaryotic cells, and play key functional roles. As such, RNase III provides an important model with which to understand mechanisms of RNA maturation, RNA decay, and gene regulation.
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PMID:Function, mechanism and regulation of bacterial ribonucleases. 1037 Oct 39

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.
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PMID:Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos. 1500 Aug 20


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