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)

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.
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PMID:Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging. 130 4

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

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.
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PMID:Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes. 876 29

We describe our initial application of a biochemical strategy, comprising combinatorial screening and rational optimization, which directly identifies oligonucleotides with maximum affinity (per unit length), specificity, and rates of hybridization to structurally preferred sites on folded RNA, to the problem of design of antisense oligonucleotides active against the hepatitis C virus (HCV). A fully randomized sequence DNA oligonucleotide (10-mer) library was equilibrated with each of two folded RNA fragments (200 and 370 nucleotides (nt)), together spanning the 5' 440 nt of an HCV transcript (by overlapping 130 nt), which were varied over a range of concentrations. The equilibrations were performed in solution under conditions determined to preserve RNA structure and to limit all RNA-DNA library oligonucleotide interactions to 1:1 stoichiometry. Subsequent Escherichia coli RNase H (endoribonuclease H: EC 3.1.26.4) cleavage analysis identified two preferred sites of highest affinity heteroduplex hybridization. The lengths and sequences of different substitute chemistry oligonucleotides complementary to these sites were rationally optimized using an iterative and quantitative analysis of binding affinity and specificity. Thus, DNA oligonucleotides that hybridized with the same affinity to the preferred sites in the folded RNA fragments found by screening as to short (< or = 25 nt) RNA complements were identified but were found to vary in length (10-18 nt) from site to site. Phosphorothioate (P=S) and 2'-fluoro (2'-F) uniformly substituted oligonucleotides also were found, which hybridized optimally to these sites, supporting the design of short (10-15-nt) and maximally specific oligonucleotides that are more nuclease-resistant (via P=S) and have higher affinity (via 2'-F) than DNA. Finally, the affinities of DNA and uniform 2'-F-, P=S-substituted 10-20-mer oligonucleotide complements for the best hybridization site, from HCV nt 355 to nt 364-374, closely corresponded to antisense mechanism inhibition activities in an in vitro translation assay and in a human cell-based HCV core protein expression assay, respectively. These results validate our strategy for the selection of hybridization-optimized and biologically active antisense oligonucleotides targeting HCV RNA and support the potential for utility in further applications.
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PMID:Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity. 899 6

Inhibition of hepatitis C virus (HCV) gene expression by antisense oligonucleotides was investigated using both a rabbit reticulocyte lysate in vitro translation assay and a transformed human hepatocyte cell expression assay. Screening of overlapping oligonucleotides complementary to the HCV 5' noncoding region and the core open reading frame (ORF) identified a region susceptible to translation inhibition between nucleotides 335 and 379. Comparison of 2'-deoxy-, 2'-O-methyl-, 2'-O-methoxyethyl-, 2'-O-propyl-, and 2'-fluoro-modified phosphodiester oligoribonucleotides demonstrated that increased translation inhibition correlated with both increased binding affinity and nuclease stability. In cell culture assays, 2'-O-methoxyethyl-modified oligonucleotides inhibited HCV core protein synthesis with comparable potency to phosphorothioate oligodeoxynucleotides. Inhibition of HCV core protein expression by 2'-modified oligonucleotides occurred by an RNase H-independent translational arrest mechanism.
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PMID:Inhibition of translation of hepatitis C virus RNA by 2-modified antisense oligonucleotides. 1035 21

Jule is the second complete long-terminal-repeat (LTR) Ty3/Gypsy retrotransposon identified to date in vertebrates. Jule, first isolated from the poeciliid fish Xiphophorus maculatus, is 4.8 kb in length, is flanked by two 202-bp LTRs, and encodes Gag (structural core protein) and Pol (protease, reverse transcriptase, RNase H, and integrase, in that order) but no envelope. There are three to four copies of Jule per haploid genome in X. maculatus. Two of them are located in a subtelomeric region of the sex chromosomes, where they are associated with the Xmrk receptor tyrosine kinase genes, of which oncogenic versions are responsible for the formation of hereditary melanoma in Xiphophorus. One almost intact copy of Jule was found in the first intron of the X-chromosomal allele of the Xmrk proto-oncogene, and a second, more corrupted copy is present only 56 nt downstream of the polyadenylation signal of the Xmrk oncogene. Jule-related elements were detected by Southern blot hybridization with less than 10 copies per haploid genome in numerous other poeciliids, as well as in more divergent fishes, including the medakafish Oryzias latipes and the tilapia Oreochromis niloticus. Database searches also identified Jule-related sequences in the zebrafish Danio rerio and in both genome project pufferfishes, Fugu rubripes and Tetraodon nigroviridis. Phylogenetic analysis revealed that Jule is the first member of the Mag family of Ty3/Gypsy retrotransposons described to date in vertebrates. This family includes the silkworm Mag and sea urchin SURL retrotransposons, as well as sequences from the nematode Caenorhabditis elegans. Additional related elements were identified in the genomes of the malaria mosquito Anopheles gambiae and the nematode Ascaris lumbricoides. Phylogeny of Mag-related elements suggested that the Mag family of retrotransposons is polyphyletic and is constituted of several ancient lineages that diverged before their host genomes more than 600 MYA.
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PMID:Jule from the fish Xiphophorus is the first complete vertebrate Ty3/Gypsy retrotransposon from the Mag family. 1115 69

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.
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PMID:Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo. 1170 13

The exon junction complex (EJC) is critical for mammalian nonsense-mediated mRNA decay and translational regulation, but the mechanism of its stable deposition on mRNA is unknown. To examine requirements for EJC deposition, we created splicing substrates containing either DNA nucleotides or RNA secondary structure in the 5' exon. Using RNase H protection, toeprinting, and coimmunoprecipitation assays, we found that EJC location shifts upstream when a stretch of DNA or RNA secondary structure appears at the canonical deposition site. These upstream shifts occur prior to exon ligation and are often accompanied by decreases in deposition efficiency. Although the EJC core protein eIF4AIII contacts four ribose 2'OH groups in crystal structures, we demonstrate that three 2'OH groups are sufficient for deposition. Thus, the site of EJC deposition is more flexible than previously appreciated and efficient deposition appears spatially limited.
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PMID:Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate. 1895 19

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position -12 (relative to the 5' splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.
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PMID:Trypanosome spliced-leader-associated RNA (SLA1) localization and implications for spliced-leader RNA biogenesis. 1902 94

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.
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PMID:Essential role of a trypanosome U4-specific Sm core protein in small nuclear ribonucleoprotein assembly and splicing. 2008 Oct 62


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