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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)

RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.
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PMID:Identification of sites of pre-MRNA/spliceosome association. 136 23

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.
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PMID:Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function. 164 89

We have isolated a 55-kDa enzyme from Saccharomyces cerevisiae on the basis of its ability to hydrolyze specifically the RNA moiety of RNA/DNA hybrids [RNase H(55)]. Remarkably, monospecific anti-[RNase H(55)] antibodies revealed that the protein associates with several small RNAs, including some of the essential yeast spliceosomal snRNAs. Moreover, immunoprecipitation as well as immunoblotting experiments demonstrated that the yeast enzyme reacts (a) with human anti-Sm autoantisera, (b) with a monoclonal antibody specific for the human snRNP proteins B/B', but (c) not with U1-ribonucleoprotein-specific autoantibodies. These results disclosed a hitherto unexpected degree of evolutionary conservation in snRNP protein structure between yeast and man. Additionally, our findings suggested a re-evaluation of the enzymatic mechanism of RNases H which recognize both RNA and RNA/DNA hybrids.
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PMID:Identification of a yeast ribonuclease H as an Sm antigen. 246 94

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.
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PMID:In vitro assembly of yeast U6 snRNP: a functional assay. 256 Jul 55

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA.DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G.C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.
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PMID:Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. 269 70

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.
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PMID:Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. 283 38

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.
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PMID:Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA. 338 87

An RNase A protection assay was employed to investigate the interaction of nuclear components with a precursor-mRNA derived from the adenovirus 2 major late transcription unit in a splicing extract from HeLa cells. Upon incubation in the extract, two regions in the precursor-RNA become resistant to digestion with RNase A. After short incubation times (5 min) at 30 degrees C, fragments mapping upstream from the branch point in the intron are obtained. After ten minutes or more, additional oligonucleotides, derived from the 5' splice site, are protected. RNase A protection of different RNA substrates demonstrates that a 5' splice site is not required for the binding of components to the branch point region. For interaction with this site, the polypyrimidine stretch just upstream from the 3' splice site is essential. Binding to the 5' splice site occurs only in the presence of an intact 3' end of the intron. Preincubation of the extract with excess unlabelled RNA containing only a 3' splice site leads to efficient competition of binding, both in the branch point region and at the 5' splice site, whereas an RNA that contains only 5'-splice-site sequences has no effect on the interaction with the mRNA precursor. This indicates that stable association with the 5' splice site requires prior binding of components in the branch point region. When splicing complexes are digested with RNase A, it becomes apparent that only the branch point region is sequestered into a ribonucleoprotein (RNP) structure in the 35 S complex. The 5' splice site becomes resistant to RNase A only when a 50 S splicing complex has been assembled. Degradation of specific regions in U1, U2 and U4 RNA with complementary oligodeoxynucleotides and RNase H has been used to analyse involvement of the U small nuclear RNPs (snRNPs) in the protection reaction. The 5' end of U2 RNA is essential for protection of the branch point region. RNA sequences in a loop of U2 RNA (nucleotides 65 to 78) are required for the formation of an RNase-A-resistant structure at the 5' splice site. Taken together, these results suggest that U2 snRNP participates in the formation of a pre-splicing complex, the 5' end of its RNA being involved in the observed binding. Conversion to a 50 S splicing complex is obtained after the binding of U1 and U4/U6 snRNPs, which also requires sequences in a loop of U2 RNA. Possible interactions between the individual snRNPs and between snRNPs and precursor-mRNA are discussed.
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PMID:Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA. 368 67

Multiple alpha- and beta-tubulin RNAs were found in the mature unfertilized eggs of the sea urchin Lytechinus pictus. The alpha-tubulin RNAs were polymorphic in number, size, and relative amounts in the eggs of different females. Five to seven different size classes [1.75-4.2 kilobases (kb)] were detected on RNA gel blots. All egg preparations contained variable amounts of 1.8- and 2.25-kb beta-tubulin RNAs, and a few of them contained an additional 2.9-kb beta-tubulin RNA. The total amount of alpha-tubulin RNA did not always parallel that of beta-tubulin RNA. A portion of all of the various alpha- and beta-tubulin RNAs were polyadenylylated. RNase H digestions ruled out the possibility that some of these RNAs represented a single transcript bearing different lengths of 3' poly(A). One class of alpha-tubulin RNAs (2.4-2.65 kb) was reduced to 2 kb by RNase H, suggesting the presence of internal oligo(A) regions. All of the egg beta-tubulin RNAs sedimented as free ribonucleoprotein particles. Only a small portion of the 1.75- to 3.6-kb alpha-tubulin RNAs, but most of the 4.2-kb alpha-tubulin RNA, were found on polysomes before fertilization. In the 30-min embryo, small amounts of each of the various alpha- and beta-tubulin RNAs were recruited onto polysomes. Thus, each of the multiple polymorphic alpha- and beta-tubulin RNAs in the egg represent translationally competent mRNA.
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PMID:Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs. 385 35

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.
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PMID:Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon. 929 34


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