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)

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.
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PMID:An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1. 912 25

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.
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PMID:SR protein and snRNP requirements for assembly of the Rous sarcoma virus negative regulator of splicing complex in vitro. 950 Oct 36

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.
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PMID:Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. 975 78

We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome.
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PMID:Initial recognition of U12-dependent introns requires both U11/5' splice-site and U12/branchpoint interactions. 1019 85

Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.
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PMID:Aberrant splicing of tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17. 1080 46

Association of U2 snRNP with the pre-mRNA branch region is the first ATP-dependent step in spliceosome assembly. The basis of this energy dependence is not known. Previously, we identified minimal intron-derived substrates that form complexes with U2 independent of ATP. Here, we identify the intron region linked to the ATP dependence of this step by comparing these substrates to longer RNAs that recapitulate the ATP requirement. This region needed to impose ATP dependence lies immediately 5' to the branch site. Sequences ranging from 6 to 14 nt yield a near linear inhibitory effect on efficiency of complex formation with U2 snRNP, with 18 nt yielding near maximal ATP dependence. This region is not protected prior to U2 addition, and RNase H targeting of the region within nuclear extract converts an ATP-dependent substrate into an ATP-independent one. Within this region, there is no sequence specificity linked with the ATP requirement, as neither a specific sequence is needed, nor even nucleobases. These data and the results of other modifications suggest models in which the 18-nt region is a target for interactions with U2 snRNP in an ATP-bound or -activated conformation.
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PMID:The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site. 1156 51

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

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

An essential step in pre-mRNA splicing is the release of the mRNA product from the spliceosome. The DEAH box RNA helicase Prp22 catalyzes mRNA release by remodeling contacts within the spliceosome that involve the U5 snRNP. Spliceosome disassembly requires a segment of more than 13 ribonucleotides downstream of the 3' splice site. I show here by site-specific crosslinking and RNase H protection that Prp22 interacts with the mRNA downstream of the exon-exon junction prior to mRNA release. The findings support a model for Prp22-catalyzed mRNA release from the spliceosome wherein a rearrangement that accompanies the second transesterification step deposits Prp22 on the mRNA downstream of the exon-exon junction. Bound to its target RNA, the 3'-->5' helicase acts to disrupt mRNA/U5 snRNP contacts, thereby liberating the mRNA from the spliceosome.
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PMID:A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. 1861 41

Mutations in two branch-point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre-mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre-mRNA splicing that mimicked pre-mRNA splicing in the patients' cells. DNA oligonucleotide-directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP-BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)-damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post-UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP-BPS interaction leading to abnormal pre-mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post-UV survival and impaired photoproduct removal.
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PMID:XPC branch-point sequence mutations disrupt U2 snRNP binding, resulting in abnormal pre-mRNA splicing in xeroderma pigmentosum patients. 1995 7


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