Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing.Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.[Key Words: Alternative splicing; RNA-binding proteins; U1 snRNP; P elements; 5' splice sites; RNA processing] Received April 16, 1992; revised version accepted May 11, 1992.Regulation of RNA splicing, the process that removes intervening sequences (introns) from mRNA precursors (pre-mRNAs) and rejoins coding sequences (exons), controls the expression of many eukaryotic genes, often according to tissue-specific or temporal cues (for reviews, see Smith et al. 1989;Green 1991;Maniatis 1991). Variations in the activities of general splicing factors may control some alternative splicing events. The general splicing factor ASF/SF2 alters 5' splice site selection in vitro and may control alternative splicing of the SV40 T/t antigen pre-mRNA (Ge and Manley 1990; Krainer et al. 1990a, b). The binding of regulatory factors to specific pre-mRNA sequences can also control alternative splicing. For example, the Sex-lethal (Sxl), transformer (tra), and tra-2 proteins appear to bind directly to specific 2present address: Department of Molecular and Cell Biology, Division of Genetics and Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720 USA.RNA target sequences to regulate a cascade of alternative splicing events in the Drosophila pathway of somatic sexual differentiation (for review, see Baker 1989). Although these and other studies provide tools for understanding mechanisms of alternative splicing, these mechanisms have remained largely speculative.The Drosophila P transposable element, which displays a simple alternative splicing pattern and is accessible to genetic and biochemical techniques, provides a valuable system to approach such mechanistic questions. The...
Nasent mRNA chains are capped at the 5' end by the addiion ofa guanylyl residue to form a G(5')ppp (5'
Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein.Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.
After infection of baby hamster kidney cells with vesicular stomatitis virus (VSV), processing and assembly of small nuclear ribonucleoproteins (snRNP) were rapidly inhibited. The Ul and U2 snRNAs accumulated as precursor species approximately 3 and 10 nucleotides longer, respectively, than the mature RNAs. Alteration in snRNP assembly was noted because the precursor snRNAs were not associated with the U-series RNA-core protein complex in infected cells. However, antibodies specific for the U2 RNA-binding protein, A', were able to precipitate pre-U2 RNAs from VSV-infected cells. These results indicated that precursors to U2 RNA were bound to A' and remained bound during virus infection. Analysis of the synthesis of proteins normally associated with Ul and U2 RNAs indicated that synthesis was unaffected at times when snRNP assembly with core proteins was blocked by the VSV. These findings suggested that the core proteins associate with one another in the absence of the snRNAs in VSV-infected cells. They further suggest a correlation between the inability of the core complex to bind the U-series snRNPs and the failure to process the 3' ends of Ul and U2 RNAs in VSV-infected cells. These effects of VSV on snRNP assembly may be related to the shutoff of host-cell macromolecular synthesis.Vesicular stomatitis virus (VSV) is a member of the negative-strand RNA virus group and is known to cause acute as well as persistent virus infections in a wide range of mammalian hosts (30). Acute infections are associated with dramatic alterations in cellular RNA metabolism and mRNA translation (31,36,40). Evidence from Wagner and coworkers (8,9,22) has implicated a short viral transcript, the leader RNA, in the shutoff of transcription by RNA polymerases (pol) II and III; however, the target of this interaction has not been elucidated. Although leader RNA is bound to the cellular La protein (15, 16) which, presumably, plays a role in synthesis or processing of RNA pol III transcripts (2,18,29), no evidence relating the leader-La association to the inhibition of cellular RNA synthesis has been found. In addition, Grinnell and Wagner (9) have shown that the leader RNA can bind to an unidentified cell protein of 65 kilodaltons (kDa), but the specificity and functional significance of this interaction is unknown.Most RNA pol II transcripts are processed via a complex series of reactions including capping, methylation, splicing, and polyadenylation (24). The small nuclear ribonucleoproteins (snRNPs) that are immunoprecipitable by the lupus Sm autoantibody specificity have been implicated in the processing of mRNA (14, 26). The snRNPs contain the snRNAs (Ul, U2, U4, U5, and U6) which are also thought to be products of RNA pol II and require trimethyl 5' capping, methylation, and excision of extraneous sequences (1,5,18,27,28,35,37). Previous work has suggested that the snRNAs are synthesized in the nucleus as precursors that are transported to the cytoplasm where they are processed to their mature sizes, assembled into RNPs, and then t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.