Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20366 (substance P)
 21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5' splice site of E4 is coincident with the selection of the 3' splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3' splice site of E4 in a manner that is clearly facilitated by a downstream 5' splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3' splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.
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PMID:U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. 128 25

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.
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PMID:Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle. 183 32

For pre-mRNAs containing multiple introns, the exon definition hypothesis has been proposed to account for the interactions that specify relatively short exons and prevent inappropriate exon-skipping [1]. Support for this hypothesis includes the finding that naturally occurring, or engineered mutations in 5' splice sites that weaken base complementary to U1 snRNA result in exon skipping due to a decrease in upstream 3' splice site activity. The reciprocal effect is also observed. For example, we found previously that the selection of the alternatively spliced rat preprotachykinin exon 4 is improved under conditions in which the adjacent 5' splice site is converted to a site with strengthened base pairing to U1 snRNA [2]. In the latter study, 3' splice site activity is improved in parallel with strengthened U1 snRNP binding to the downstream 5' splice site. Subsequent RNA-protein crosslinking experiments have provided evidence for exon bridging interactions between U2AF bound to the 3' splice site and U1 snRNP bound to the downstream 5' splice site in the preprotachykinin substrates [3]; see Figure 1. U2AF, a polypyrimidine tract binding protein composed of 65 and 35 kD subunits, is required for U2 snRNP binding to the adjacent branch site [4], [5]. In this work we have reconstituted exon bridging activity with purified components. These results show that U1 snRNP in addition to U2AF are the two components required to reconstitute full activity in vitro. The purified system has been used to test variants of U2AF and U1 snRNP. Our results show that the U1-A and U1-C proteins are dispensable for exon bridging activity. In addition, the 35 kD subunit of U2AF appears to be dispensable, at least under certain conditions.
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PMID:Reconstitution of exon-bridging activity with purified U2AF and U1 snRNP components. 864 75