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1.
Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins which stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. We have tested this model by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) bound equally to the pre-mRNA in the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa bound prior to most SR proteins and hTra2beta and its level of binding did not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA was, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract. 相似文献
2.
Vela E Hilari JM Roca X Muñoz-Mármol AM Ariza A Isamat M 《RNA (New York, N.Y.)》2007,13(12):2312-2323
The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3. 相似文献
3.
Linking C5 deficiency to an exonic splicing enhancer mutation 总被引:6,自引:0,他引:6
Pfarr N Prawitt D Kirschfink M Schroff C Knuf M Habermehl P Mannhardt W Zepp F Fairbrother WG Fairbrother W Loos M Burge CB Pohlenz J 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(7):4172-4177
As an important component of the innate immune system, complement provides the initial response to prevent infections by pathogenic microorganisms. Patients with dysfunction of C5 display a propensity for severe recurrent infections. In this study, we present a patient with C5 deficiency demonstrated by immunochemical and functional analyses. Direct sequencing of all C5 exons displayed no mutation of obvious functional significance, except for an A to G transition in exon 10 predicting an exchange from lysine to arginine. This sequence alteration was present in only one allele of family members with a reduced serum C5 concentration and in both alleles of the patient with almost complete C5 deficiency, suggesting that this alteration may be producing the phenotype. Recent findings indicate that distinct nucleotide sequences, termed exonic splicing enhancers (ESEs), influence the splicing process. cDNA from all family members harboring the mutated allele showed skipping of exon 10, which resulted in a premature STOP codon, explaining the lack of C5 in the propositus. Sequence analysis of the mutated region revealed the substitution to be located within an ESE, as predicted by the RESCUE-ESE program. The altered ESE sequence is located close to the 5' splicing site and also lowers the predicted strength of the splice site itself. This apparently inconsequential sequence alteration represents a noncanonical splicing mutation altering an ESE. Our finding sheds a new light on the role of putative silent/conservative mutations in disease-associated genes. 相似文献
4.
5.
6.
Localization of an exonic splicing
enhancer responsible for mammalian natural trans-splicing 总被引:4,自引:0,他引:4 下载免费PDF全文
Concha Caudevilla Carles Codony Dolors Serra Guillem Plasencia Ruth Romn Adolf Graessmann Guillermina Asins Montserrat Bach-Elias Fausto G. Hegardt 《Nucleic acids research》2001,29(14):3108-3115
7.
A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. 总被引:20,自引:14,他引:20 下载免费PDF全文
J Ramchatesingh A M Zahler K M Neugebauer M B Roth T A Cooper 《Molecular and cellular biology》1995,15(9):4898-4907
The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion. 相似文献
8.
Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences. 相似文献
9.
Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55. 总被引:1,自引:2,他引:1 下载免费PDF全文
Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA. 相似文献
10.
Background
The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid. 相似文献11.
The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream. 总被引:11,自引:3,他引:11 下载免费PDF全文
The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds. 相似文献
12.
Repression of alpha-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract 下载免费PDF全文
Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the alpha-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the alpha-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 3' end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF(65) binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF(65), thereby specifically blocking splicing of the SM exon. 相似文献
13.
Arrisi-Mercado P Romano M Muro AF Baralle FE 《The Journal of biological chemistry》2004,279(38):39331-39339
Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site. 相似文献
14.
Zhiqi Chen Xuezhong Ma Jianhua Zhang Jim Hu Reginald M. Gorczynski 《Nucleic acids research》2010,38(19):6684-6696
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. 相似文献
15.
The dystrophin gene is composed of at least 86 exons and the occurrence of several alternative splicing sites, mainly occurring in its 3' region, is a well recognised phenomenon. We have found that exon 4 can also be alternatively spliced in human skeletal and cardiac muscle. 相似文献
16.
Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta 总被引:10,自引:0,他引:10
Jiang Z Tang H Havlioglu N Zhang X Stamm S Yan R Wu JY 《The Journal of biological chemistry》2003,278(21):18997-19007
Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2 beta protein. The interaction between Tra2 beta and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2 beta protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2 beta, suggesting that Tra2 beta may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies. 相似文献
17.
Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation. 相似文献
18.
Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing. 相似文献
19.
The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays. 相似文献
20.
Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing. 总被引:1,自引:6,他引:1 下载免费PDF全文
We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors. 相似文献