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 identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F. Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle. All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts. U5E and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species. U5D, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, U5DL, and a shorter species, U5DS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae. We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, U5E and U5F can be specifically and completely degraded in splicing extracts by oligonucleotide-directed RNase H cleavage. All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/U5/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes. The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.
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PMID:Three novel functional variants of human U5 small nuclear RNA. 131 Jan 51

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.
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PMID:Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP. 165 32

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.
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PMID:The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly. 185 Jan 27

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.
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PMID:U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. 255 94

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.
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PMID:U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. 299 75

A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP) fractions, which showed binding activities for both 5' and 3' splice site RNAs, were studied for the sequence specificity of their binding. Results indicate that the U1-rich snRNP fraction can recognize both 5' and 3' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5' splice site sequences, but not to a consensus 3' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5' splice site, but not a 3' splice site. The binding activity for the 5' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60 degrees C. The binding activity to a 3' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.
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PMID:Recognition of 5' and 3' splice site sequences in pre-mRNA studied with a filter binding technique. 304 Jul 11

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.
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PMID:Translational regulation and deadenylation of a protamine mRNA during spermiogenesis in the mouse. 646 65

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.
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PMID:Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry. 753 31

The hyperthermophilic archaeon Sulfolobus acidocaldarius uses a novel RNA-containing endonuclease to excise and mature 16S rRNA from the precursor (pre) rRNA transcript. A cell-free processing system has been developed using an in vitro transcribed RNA substrate containing the entire 144 nucleotide 5' external transcribed spacer (5'ETS) and the first 72 nucleotides of 16S rRNA. The cell-free extract cleaves in the 5'ETS at positions -99, -31, and +1 (i.e., the 5'ETS-16S junction). These positions are at or near the positions cleaved in vivo during processing of the pre rRNA transcript. The processing activity has been purified between 100 and 200-fold and appears to contain five or six polypeptide components and perhaps as many as 10 different small RNA components. Using combined reverse transcription-PCR amplification, full or partial cDNA copies of two of the RNA components have been obtained. One of the RNAs exhibits sequence and structural similarities to eukaryotic U3 snoRNA. The processing activity has been shown to be inactivated by micrococcal nuclease. It can be reactivated by reconstituting using bulk RNA from S.acidocaldarius but not bulk RNA from distantly related organisms. The activity is also abolished by RNase H digestion in the presence of oligonucleotides complementary to the U3-like RNA. These results demonstrate that the U3-like RNA is an essential component of the pre rRNA processing RNP endonuclease. Furthermore, this RNP endonuclease is not a derived eukaryotic feature, instead its existence predates the divergence of archaea and eukaryotes.
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PMID:Preribosomal RNA processing in archaea: characterization of the RNP endonuclease mediated processing of precursor 16S rRNA in the thermoacidophile Sulfolobus acidocaldarius. 872 97

Oligodesoxyribonucleotide-directed cleavage of protein-deficient Thermus thermophilus derivatives of the 30S ribosomal subunits with RNase H is described. A homogeneous RNP fragment has been isolated as a result of the cleavage and subsequent purification in the sucrose gradient. It corresponds to the central and 5' domains of the 30S ribosomal subunit. The high compactness of the fragment in solution suggests that it can be considered as a 'beheaded' derivative of the 30S ribosomal subunit. The absence of a reconstitution stage in isolation of the 22S RNP fragment provides for its preparation in large amounts.
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PMID:Preparation of a 'beheaded' derivative of the 30S ribosomal subunit. 945 54


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