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:3.1.13.1 (exoribonuclease)
732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
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PMID:Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation. 5 Sep 2

An exoribonuclease that hydrolyzes single-stranded RNA by a 5'----3' mode yielding 5'-mononucleotides has been purified from human placental nuclei. Chromatographic studies of crude placental nuclear extracts suggest that the enzyme is a relatively abundant nuclear RNase. Poly(A) is degraded by a processive mechanism while rRNA is degraded in a partially non-processive manner, possibly because of its secondary structure. The enzyme has an apparent molecular weight of 113,000, derived from determinations of the Stokes radius (43 A) and sedimentation coefficient (6.3 S). Substrates with 5'-phosphomonoester end groups are 10-20 times better than 5'-dephosphorylated substrates. The locale of the enzyme in nuclei of normal human cells as well as its mode of action suggest a role in nuclear RNA processing or turnover.
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PMID:A 5'----3' exoribonuclease of human placental nuclei: purification and substrate specificity. 243 25

Highly purified and commercially available preparations of reverse transcriptases from retroviruses contain a 3' to 5' exoribonuclease activity capable of hydrolyzing synthetic homopolyribonucleotides having a 3'-OH end. The exoribonuclease activity of reverse transcriptase preparations from Rous associated virus-2 was further characterized. This exoribonuclease activity cleaves poly(C) and poly(U) exonucleolytically from the 3'-OH end to produce nucleoside 5'-phosphates. Poly(A), poly(G), circular polyribonucleotide, and double-stranded polyribonucleotide were not hydrolyzed by the activity. This is a novel type of exoribonuclease activity.
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PMID:Exoribonuclease activity of purified reverse transcriptase preparations from retroviruses. 247 86

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.
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PMID:Polyriboadenylate sequences at the 3'-termini of ribonucleic acid obtained from mammalian leukemia and sarcoma viruses. 437 12

The rpsO mRNA, encoding ribosomal protein S15, is only partly stabilized when the three ribonucleases implicated in its degradation--RNase E, polynucleotide phosphorylase, and RNase II--are inactivated. In the strain deficient for RNase E and 3'-to-5' exoribonucleases, degradation of this mRNA is correlated with the appearance of posttranscriptionally elongated molecules. We report that these elongated mRNAs harbor poly(A) tails, most of which are fused downstream of the 3'-terminal hairpin at the site where transcription terminates. Poly(A) tails are shorter in strains containing 3'-to-5' exoribonucleases. Inactivation of poly(A) polymerase I (pcnB) prevents polyadenylylation and stabilizes the rpsO mRNA if RNase E is inactive. In contrast polyadenylylation does not significantly modify the stability of rpsO mRNA undergoing RNase E-mediated degradation.
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PMID:Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli. 773 15

Ribonuclease II is a processive 3' exoribonuclease in Escherichia coli. It degraded substrates with 3'-OH or 2',3'-cyclicP ends slightly faster than those with 3'-P or 2'-P groups with a turnover number of approximately 70 nt/s at 37 degrees C. RNase II does not degrade DNA but the specificity for ribose was not for the cleavage bond but rather for ribo-bonds three to four nucleotides (nt) upstream, which could explain why the limit digest is a dimer. Oligonucleotides (oligos) of deoxy(C) were reversible competitive inhibitors of the enzyme and indicated a strong upstream binding site (approximately 15 to 27 nt from the 3' end). These oligos could protect RNase II from inactivation by heat or from diethylpyrocarbonate, an agent that preferentially reacts with His residues. Compared to oligo(dC), oligos of (dA) were at least 500 times less effective inhibitors of RNase II. Using mixed oligo(dAdC) inhibitors, an obligatory 3' to 5' direction of binding into the catalytic site was shown. From the reaction kinetics of RNase II under different conditions it was concluded that the enzyme recognition differs for poly(A), poly(C) and poly(U). Poly(C) was degraded more slowly than poly(A) or poly(U) with a 3.5 times slower Vmax, while rate differences between small oligos were extreme; oligo(A)7 was degraded > 100 times faster than oligo(C)7. Ethanol, which weakens hydrophobic interactions, increased the reaction velocity of poly(C) to that of poly(A) and poly(U). It had no effect on the reaction velocities of poly(A) or poly(U), but decreased the binding of poly(A) markedly. Oligo(A) was bound more strongly to a hydrophobic column than was oligo(C). Salt, which affects charge interactions, decreased the binding affinity and/or association rate of poly(C) to RNase II, had a lesser effect on poly(U), but the reactions of poly(A) were unaffected even in much higher concentrations of salt. A clue to the slower reaction velocity of poly(C) was shown when the reaction intermediates were viewed by PAGE. At lower temperatures of reaction (< 25 degrees C), there were more intense bands separated by discrete distances of approximately 12 nt during the degradation of poly(C) by RNase II. Chase experiments showed that these stops were accounted for by dissociation of poly(C) from the enzyme. They were not seen when poly(C) was degraded at 37 degrees C or degraded in the presence of 20% ethanol at any temperatures, nor were they seen when poly(A) or poly(U) was degraded even at low temperatures.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The processive reaction mechanism of ribonuclease II. 796 9

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 rpsO mRNA of E. coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I. We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E. It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II. A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2. Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells. Poly(A)polymerase I contributes differently to the instability of these fragments. The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript. We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA.
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PMID:E. coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent degradation of the mRNA. 1004 80

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.
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PMID:Cap-dependent deadenylation of mRNA. 1069 48

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single-stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3' termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.
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PMID:Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. 1084 84


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