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1.
Biochemical purification of a pre-mRNA splicing activity from HeLa cells that stimulates distal alternative 3' splice sites in a concentration-dependent manner resulted in the identification of RNPS1, a novel general activator of pre-mRNA splicing. RNPS1 cDNAs, encoding a putative nucleic-acid-binding protein of unknown function, were previously identified in mouse and human. RNPS1 is conserved in metazoans and has an RNA-recognition motif preceded by an extensive serine-rich domain. Recombinant human RNPS1 expressed in baculovirus functionally synergizes with SR proteins and strongly activates splicing of both constitutively and alternatively spliced pre-mRNAs. We conclude that RNPS1 is not only a potential regulator of alternative splicing but may also play a more fundamental role as a general activator of pre-mRNA splicing.  相似文献   

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The multiprotein exon junction complex (EJC) is deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. In mammalian cells, this complex serves as a key modulator of spliced mRNA metabolism. To date, neither the complete composition nor the exact assembly pathway of the EJC has been entirely elucidated. Using in vitro splicing and a two-step chromatography procedure, we have purified the EJC and analyzed its components by mass spectrometry. In addition to finding most of the known EJC factors, we identified two novel EJC components, Acinus and SAP18. Heterokaryon analysis revealed that SAP18 is a shuttling protein whereas Acinus is restricted to the nucleus. In MS2 tethering assays Acinus stimulated gene expression at the RNA level, while MLN51, another EJC factor, stimulated mRNA translational efficiency. Using tandem affinity purification (TAP) of proteins overexpressed in HeLa cells, we demonstrated that Acinus binds directly to another EJC component, RNPS1, while stable association of SAP18 to form the trimeric apoptosis and splicing associated protein (ASAP) complex requires both Acinus and RNPS1. Using the same methodology, we further identified what appears to be the minimal stable EJC core, a heterotetrameric complex consisting of eIF4AIII, Magoh, Y14, and MLN51.  相似文献   

4.
Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.  相似文献   

5.
Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.  相似文献   

6.
New information about the pathway of eukaryotic gene expression indicates that many of the steps in this pathway are functionally interconnected. An important link has recently emerged between pre-mRNA splicing and the post-splicing events such as mRNA export and mRNA decay. Recent results reveal that the coupling is mediated by a novel group of nuclear mRNA-binding proteins that are recruited to the mRNAs by spiceosome. These proteins, including Y14, Aly/REF, RNPS1, SRm160, and DEK, are assembled into a stable complex near exon-exon junctions of spliced mRNAs. Several of them persist in their attachment to mRNAs in the cytoplasm thus communicating the history of splicing to the downstream events. The detailed mechanism of coupling and the factors that mediate these processes remain to be determined in the coming years.  相似文献   

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Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.  相似文献   

9.
Over the past decade many studies have revealed a complex web of interconnections between the numerous steps required for eukaryotic gene expression. One set of interconnections link nuclear pre-mRNA splicing and the subsequent metabolism of the spliced mRNAs. It is now apparent that the means of connection is a set of proteins, collectively called the exon junction complex, which are deposited as a consequence of splicing upstream of mRNA exon-exon junctions.  相似文献   

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Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.  相似文献   

12.
Identification of a regulated pathway for nuclear pre-mRNA turnover   总被引:31,自引:0,他引:31  
We have identified a nuclear pathway that rapidly degrades unspliced pre-mRNAs in yeast. This involves 3'-->5' degradation by the exosome complex and 5'-->3' degradation by the exonuclease Rat1p. 3'-->5' degradation is normally the major pathway and is regulated in response to carbon source. Inhibition of pre-mRNA degradation resulted in increased levels of pre-mRNAs and spliced mRNAs. When splicing was inhibited by mutation of a splicing factor, inhibition of turnover resulted in 20- to 50-fold accumulation of pre-mRNAs, accompanied by increased mRNA production. Splicing of a reporter construct with a 3' splice site mutation was also increased on inhibition of turnover, showing competition between degradation and splicing. We propose that nuclear pre-mRNA turnover represents a novel step in the regulation of gene expression.  相似文献   

13.
The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.  相似文献   

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We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. When assembled in vitro, this so-called 'exon-exon junction complex' (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm.  相似文献   

16.
V Gerke  J A Steitz 《Cell》1986,47(6):973-984
A HeLa cell nuclear extract active in splicing of pre-mRNA has been fractionated to identify the component that interacts with the 3' splice site. The activity that binds this region in an RNAase T1 protection assay copurifies with a 70 kd protein which is recognized by anti-Sm antibodies. Protein blots probed with labeled mRNA precursors either containing or lacking an intact 3' splice site reveal that the 70 kd polypeptide can bind pre-mRNA after immobilization on nitrocellulose and that it shows a preference for sequences located between the 3' splice junction and the site of lariat formation. Cofractionation during chromatography and immunoprecipitation by anti-2,2,7-trimethylguanosine antibodies demonstrate that the 3' splice site binding component associates with small nuclear ribonucleoprotein particles in low (1 mM) but not high (15 mM) Mg++ concentrations.  相似文献   

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BACKGROUND: Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins. RESULTS: We describe the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel beta sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface. CONCLUSIONS: The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions.  相似文献   

19.
The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.  相似文献   

20.
Pre-mRNA splicing removes introns and leaves in its wake a multiprotein complex near the exon-exon junctions of mRNAs. This complex, termed the exon-exon junction complex (EJC), contains at least seven proteins and provides a link between pre-mRNA splicing and downstream events, including transport, localization, and nonsense-mediated mRNA decay. Using a simple whole cell lysate system we developed for in vitro splicing, we prepared lysates from cells transfected with tagged EJC proteins and studied the association of these proteins with pre-mRNA, splicing intermediates, and mRNA, as well as formation of the EJC during splicing. Three of the EJC components, Aly/REF, RNPS1, and SRm160, are found on pre-mRNA by the time the spliceosome is formed, whereas Upf3b associates with splicing intermediates during or immediately after the first catalytic step of the splicing reaction (cleavage of exon 1 and intron-lariat formation). In contrast, Y14 and magoh, which remain stably associated with mRNA after export to the cytoplasm, join the EJC during or after completion of exon-exon ligation. These findings indicate that EJC formation is an ordered pathway that involves stepwise association of components and is coupled to specific intermediates of the splicing reaction.  相似文献   

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