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:3.1.26.4 (RNase H)
2,751 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

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.
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PMID:Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation. 247 10

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.
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PMID:Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors. 364 91

The poly(A) sequence of 30 to 40S Rous sarcoma virus RNA, prepared by digestion of the RNA with RNase T(1), showed a rather homogenous electrophoretic distribution in formamide-polyacrylamide gels. Its size was estimated to be about 200 AMP residues. The poly(A) appears to be located at or near the 3' end of the 30 to 40S RNA because: (i) it contained one adenosine per 180 AMP residues, and because (ii) incubation of 30 to 40S RNA with bacterial RNase H in the presence of poly(dT) removed its poly(A) without significantly affecting its hydrodynamic or electrophoretic properties in denaturing solvents. The viral 60 to 70S RNA complex was found to consist of 30 to 40S subunits both with (65%) and without (approximately 30%) poly(A). The heteropolymeric sequences of these two species of 30 to 40S subunits have the same RNase T(1)-resistant oligonucleotide composition. Some, perhaps all, RNase T(1)-resistant oligonucleotides of 30 to 40S Rous sarcoma virus RNA appear to have a unique location relative to the poly(A) sequence, because the complexity of poly(A)-tagged fragments of 30 to 40S RNA decreased with decreasing size of the fragment. Two RNase T(1)-resistant oligonucleotides which distinguish sarcoma virus Prague B RNA from that of a transformation-defective deletion mutant of the same virus appear to be associated with an 11S poly(A)-tagged fragment of Prague B RNA. Thus RNA sequences concerned with cell transformation seem to be located within 5 to 10% of the 3' terminus of Prague B RNA.
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PMID:Properties and location of poly(A) in Rous sarcoma virus RNA. 437 9

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.
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PMID:Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis. 633 Jun 82

Region 980-1061 in human 18S rRNA was chosen on the basis of our previous results indicating, that the cross-linking sites of alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their ability to bind selectively at the desired rRNA sequences was proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Only four of the probes were able to bind to 40S subunits indicating, that the corresponding 18S rRNA sequences 980-994, 987-1001, 1025-1039 and 1032-1046 are exposed within the subunits. None of the probes inhibited tRNA-dependent binding of oligo(U) messengers to 40S subunits. Nevertheless, two probes (complementary to 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA.eIF-2.GTP was strongly affected by the same oligomers. Thus 987-1001 and 1025-1039 18S rRNA sequences are supposed to be involved in interaction with mRNA in the course of translation.
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PMID:[Functional value of 980-1061 sequences of human 18S ribosomal RNA using complementary DNA probes]. 772 52

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.
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PMID:Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits. 871 1

2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.
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PMID:A new method for detecting sites of 2'-O-methylation in RNA molecules. 929 93

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.
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PMID:Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes. 906 Oct 30

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.
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PMID:Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. 1199 38


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