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 recently developed antisense splicing assay was used to determine the relative activities of 2'-O-methoxyethoxy (2'-MOE) phosphorothioate oligonucleotides containing base modifications. In the assay, RNase H-inactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an Enhanced Green Fluorescence Protein (EGFP) reporter pre-mRNA stably expressed in HeLa cells. Thus, the extent of EGFP upregulation is proportional to the antisense activity of the tested molecule. The base modifications included C-5 propynyl analogs of uridine and cytidine and phenoxazine and G-clamp analogs of cytosine. Base-modified 2'-MOE oligonucleotides were delivered to the HeLa EGFP-654 test cells by cationic lipid transfection or scrape-loading or without any delivery method (free uptake). When delivered with a cationic lipid, the G-clamp and phenoxazine oligomers showed increases in activity over the unmodified 2'-MOE parent compound. However, when delivered by scrape-loading or without a delivery method, the unmodified oligomer performed best. The results suggest that base modifications do not enhance the free uptake activity of RNase H inactive 2'-MOE oligomers.
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PMID:Effects of base modifications on antisense properties of 2'-O-methoxyethyl and PNA oligonucleotides. 1295 12

Virtually all uridines in the branch site recognition region (BSRR) of vertebrate U2 are converted into pseudouridines after initial transcription. Here, we report a functional analysis of these modified nucleotides using the Xenopus oocyte reconstitution system. Using site-specific (32)P-labeling and TLC, we show that U2 pseudouridylation occurs much faster in the BSRR than in the 5'-terminal region. To functionally dissect the pseudouridines in the BSRR, we replaced each uridine with 5-fluorouridine (unmodifiable nucleotide) using site-specific RNase H cleavage directed by 2'-O-methyl-RNA-DNA chimeras followed by three-piece ligation. Whereas in vitro transcribed U2 containing no 5-fluorouridines rescued splicing in U2-depleted oocytes, no rescue was observed with U2 RNA containing 5-fluorouridines introduced into the BSRR. Additionally, U2 RNA containing 5-fluorouridines in the BSRR specifically inhibited pseudouridylation in the BSRR of in vitro transcribed U2 injected at a later time, although pseudouridylation in the 5'-end region was not affected. Our reconstitution results indicated that prior injection into U2-depleted oocytes with U2 RNA containing 5-fluorouridines in the BSRR almost completely abrogated the ability of in vitro transcribed U2 to rescue splicing, whereas full rescue was obtained with either cellular U2 or U2 containing pseudouridines in the BSRR. Further analyses using glycerol-gradient and native gel electrophoresis indicated that U2 RNAs lacking the BSRR pseudouridines do not participate in the assembly of the functionally active 17S U2 snRNP and the spliceosome. We conclude that the BSRR pseudouridines of vertebrate U2 are required for complete snRNP assembly and pre-mRNA splicing in Xenopus oocytes.
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PMID:Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes. 1503 77

Post-transcriptional modifications were mapped in domains II, IV and V of 23S RNA from the archaeon Haloarcula marismortui. The RNA was investigated by two primer extension techniques using reverse transcriptase and three mass spectrometry techniques. One primer extension technique utilized decreasing concentrations of deoxynucleotide triphosphates to detect 2'-O-ribose methylations and other polymerase blocking modifications. In the other, the rRNA was chemically modified, followed by mild alkaline hydrolysis to map pseudo-uridine groups (Psis). RNA fragments for mass spectrometry were isolated from 23S rRNA by site-directed RNase H or mung bean nuclease digestion followed by gel purification. Modified RNase digestion fragments were identified with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the modifications were further studied by tandem MS. Psis suggested by the primer extension technique were verified by specific cyanoethylation and mass spectrometric detection. A total of only five post-transcriptionally methylated nucleotides and three Psis were detected in the three 23S rRNA domains. One of the methylated nucleotides has not been reported while a dispute about the number of Psis is solved. The limited number of modified nucleotides suggests that H. marismortui does not have special needs for extensive rRNA modifications. We have performed detailed investigations on the three-dimensional location and molecular interactions of the modified nucleotides by computer analysis. Our results show that all the modified positions are at regions with RNA-RNA contacts and all except one are at the surface of the subunit and in functionally important regions.
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PMID:The archaeon Haloarcula marismortui has few modifications in the central parts of its 23S ribosomal RNA. 1582 54

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV.
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PMID:RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase. 2334 89


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