Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The in vitro interaction of recombinant hnRNP A1 with purified snRNPs and with pre-mRNAs was investigated. We show that protein A1 can stably bind U2 and U4 snRNP but not U1. Oligo-RNAse H cleavage of U2 nucleotides involved in base pairing with the branch site, totally eliminates the A1-U2 interaction. RNase T1 protection and immunoprecipitation experiments demonstrate that recombinant protein A1 specifically binds the 3'-end regions of both beta-globin and Ad-2 introns. However, while on the beta-globin intron only binding to the polypyrimidine tract was observed, on the Ad-2 intron a 32 nt fragment encompassing the branch point and the AG splice-site dinucleotide was bound and protected. Such protection was drastically reduced in the presence of U2 snRNP. Altogether these results indicate that protein A1 can establish a different pattern of association with different pre-mRNAs and support the hypothesis that this protein could play a role in the annealing of U2 to the branch site and hence in the early events of pre-splicing complex assembly.
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PMID:Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly. 132 35

We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA.
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PMID:3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei. 153 84

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

We investigated the interaction of U2 snRNP with the branch-3' splice site region of three human beta-globin pre-mRNAs carrying nearly complete (BamHI RNA), 24 nt (Avall RNA) and 14 nt (Accl RNA) of exon 2. All supported splicing, but mRNAs yields were respectively 2 and 10 times lower for Avall and Accl RNAs than for BamHI. Analysis of RNase T1-resistant fragments immunoprecipitated by an anti-(U2)RNP antibody at early times of the splicing reaction showed that the protection encompasses both the branch point region and the end of the intron in BamHI and Avall, but essentially only the branch point in Accl RNAs. Later on, this protection becomes less detectable in BamHI, is reinforced in Avall and remains poorly detectable in Accl RNAs. Similar experiments performed at late times with an anti-Sm antibody recognizing all snRNPs showed that the end of the intron is protected in all but BamHI RNAs. These results support the conclusion that U2 snRNP binds to a fully efficient precursor (BamHI RNA) through another factor(s) recognizing the 3' splice site (U5 snRNP and the so-called U2AF protein are likely candidates). Either the absence of an initial contact between U2 snRNP and the factor(s) recognizing the end of the intron (Accl RNA) or the unability of this ternary complex to undergo a conformational change (Avall RNA) could render these severely truncated precursors poor substrates. These different situations have consequences on the branch point selection itself. BamHI and Avall RNAs use three functional branch points at early times, the usual A residue at -37 and two U residues at -17 and -22. Accl RNA uses only one branch point at -37. Later on, all three branch points are used at the same rate in Avall, while the usual one prevails in BamHI RNAs.
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PMID:Interplay between U2 snRNP and 3' splice factor(s) for branch point selection on human beta-globin pre-mRNA. 213 8

As demonstrated by RNase T1 protection assays at 0 degrees C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloride bind to the 5' and 3' splice sites of human beta-globin pre-mRNA, respectively. We also devised a saturation-complementation assay and have found that this purified U5 snRNP, unlike U1, successfully competes with snRNP-free fractions of nuclear proteins which inhibit spliceosome assembly and splicing. Restoration of activity requires intact U5 snRNA and correlates with the presence of the 100 Kd intron binding protein (IBP) which we have previously characterized (Tazi et al., 1986, Cell 47, 755-766). Our results are compatible with a model in which the recognition of the 3' splice site by IBP-U5 snRNP is one of the earliest events of the spliceosome assembly. It could organize the structure of the 3' splice site region of the human beta-globin like pre-mRNAs. However, on the basis of results showing that beta-globin and major late adenovirus seem to have different requirements with respect to IBP-U5 snRNP, it appears that some pre-mRNAs could have a native structure that necessitates less if at all IBP-U5.
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PMID:Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assembly and splicing by snRNP-free nuclear proteins. 252 52

rp5l B pre-mRNA, like many Saccharomyces cerevisiae primary transcripts, contains a secondary structure within its intron sequence. The structure is required for optimal in vivo splicing efficiency and includes two complementary regions near the 5' splice site and the branchpoint (UB1 and DB1, respectively). An intron-containing RNA was probed in vitro with RNase T1 and dimethyl sulfate (DMS), and is folded as expected. We have also examined in vitro splicing of rp5l B pre-mRNA, by analyzing the formation of splicing complexes as well as splicing products. Similar analyses were done with mutant pre-mRNAs containing shortened or lengthened UB1/DB1 base pairing regions. Our experiments indicate that the secondary structure acts at an early step of spliceosome assembly to aid the formation of U1 snRNP-containing commitment complexes. pre-mRNAs were probed with DMS in vivo and the folding takes place inside cells. The effect of the different UB1/DB1 interactions on in vivo splicing efficiency was also analyzed. The results are consistent with the idea that the intramolecular interaction takes place prior to or at the beginning of spliceosome assembly.
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PMID:Intramolecular structure in yeast introns aids the early steps of in vitro spliceosome assembly. 871 81

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