Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 75
Filter
1.
Hum Mutat ; 28(6): 599-612, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17311297

ABSTRACT

We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.


Subject(s)
Alternative Splicing/genetics , Genes, Neurofibromatosis 1 , Mutation , Neurofibromatosis 1/genetics , RNA Splice Sites/genetics , Adult , Austria , Child , Cohort Studies , Computer Simulation , DNA Mutational Analysis , Exons , Humans , Neurofibromatosis 1/diagnosis , Sensitivity and Specificity , Sequence Deletion
2.
J Virol ; 75(18): 8487-97, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11507194

ABSTRACT

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.


Subject(s)
3' Untranslated Regions , Exons , Gene Silencing , HIV-1/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , RNA Splicing , RNA, Viral , Ribonucleoproteins/metabolism , 5' Untranslated Regions , Binding Sites , Consensus Sequence , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Mutagenesis
3.
Curr Opin Cell Biol ; 13(3): 302-9, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11343900

ABSTRACT

The past year has witnessed refinements in models of spliceosome assembly pathways and in the understanding of how splicing factors of the serine/arginine-rich (SR) protein family function. The role of splicing in human genetic diseases has also received a lot of attention recently as exonic splicing enhancers become better understood.


Subject(s)
Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA Splice Sites/physiology , RNA Splicing/physiology , Spliceosomes/metabolism , Animals , Biological Therapy/methods , Genetic Variation/genetics , Humans , Models, Molecular , Nuclear Proteins/genetics , RNA Precursors/genetics , RNA Splice Sites/genetics , RNA Splicing/genetics , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Spliceosomes/genetics
4.
RNA ; 7(3): 471-82, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11333026

ABSTRACT

SR proteins play critical roles in the major pre-mRNA splicing pathway. A second pathway processes U12-dependent AT-AC introns. We demonstrate, by biochemical complementation, the requirement for SR proteins in splicing of AT-AC introns. Whereas SR proteins were sufficient to activate splicing of a P120 AT-AC intron, splicing of a sodium channel AT-AC intron required an additional nuclear fraction. Individual recombinant SR proteins promoted splicing of both substrates, but displayed marked preferences. SR proteins supported basal AT-AC splicing, and also splicing stimulation via a downstream enhancer or conventional 5' splice site. Analysis of chimeric transcripts revealed that information dispersed throughout exons and introns dictates SR protein specificity and the requirement for the additional nuclear fraction. Thus, SR proteins function in both major and minor splicing pathways, and in coordinating the activities of both spliceosomes via exon definition. These results suggest that despite the substantial differences in intron consensus sequences and in four of the five snRNPs in each spliceosome, at least some of the interactions involving SR proteins are conserved between the two pathways.


Subject(s)
Exons , Introns , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA Precursors/metabolism , RNA Splicing , RNA, Messenger/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Cell-Free System , HeLa Cells , Humans , NAV1.4 Voltage-Gated Sodium Channel , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Sodium Channels/genetics
5.
EMBO J ; 20(4): 864-71, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179230

ABSTRACT

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.


Subject(s)
Adenovirus E4 Proteins/metabolism , Neoplasm Proteins/metabolism , RNA Splicing , HeLa Cells , Humans , Open Reading Frames , Phosphorylation , Protein Binding
6.
Nat Genet ; 27(1): 55-8, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11137998

ABSTRACT

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.


Subject(s)
Codon, Nonsense/genetics , Exons/genetics , Genes, BRCA1/genetics , Mutation, Missense/genetics , RNA Splicing/genetics , Amino Acid Motifs , Amino Acid Substitution/genetics , BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Base Sequence , Enhancer Elements, Genetic/genetics , Humans , Molecular Sequence Data , Nuclear Proteins/chemistry , Open Reading Frames/genetics , Phenotype , Phosphoproteins/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins , Serine-Arginine Splicing Factors
7.
Mol Cell ; 8(6): 1351-61, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11779509

ABSTRACT

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.


Subject(s)
Exons/genetics , Gene Silencing , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , RNA Splicing/genetics , Regulatory Sequences, Nucleic Acid/genetics , Ribonucleoproteins/antagonists & inhibitors , Base Sequence , Binding Sites , Cell Extracts , Gene Products, tat/genetics , Genetic Complementation Test , Glycine/metabolism , HIV-1/genetics , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Kinetics , Models, Genetic , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA-Binding Proteins , Ribonucleoproteins/chemistry , Ribonucleoproteins/deficiency , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors , Substrate Specificity , tat Gene Products, Human Immunodeficiency Virus
8.
Genes Dev ; 14(24): 3166-78, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11124808

ABSTRACT

SR proteins are essential pre-mRNA splicing factors that act at the earliest stages of splice-site recognition and spliceosome assembly, as well as later in the splicing pathway. SR proteins consist of one or two RNA-recognition motifs and a characteristic arginine/serine-rich C-terminal RS domain. The RS domain, which is extensively phosphorylated, mediates the subcellular localization of individual SR proteins and also functions as a splicing activation module, apparently by engaging in protein-protein interactions. The RS domain of SF2/ASF is dispensable for the concentration-dependent effects of this SR protein on alternative splice-site selection. However, this RS domain is highly conserved phylogenetically, and was shown to be required for constitutive splicing in vitro and for cell viability. Here, we demonstrate that the RS domain of SF2/ASF is, in fact, dispensable for splicing of several substrates, including constitutive and enhancer-dependent pre-mRNAs. The requirement for this RS domain is substrate specific, and correlates with the strength of the splicing signals. When the 3' splice site is weak, both the SF2/ASF RS domain and U2AF(35) are required for splicing. These results show the existence of an RS domain-independent function of SR proteins in constitutive and enhancer-dependent splicing, and suggest mechanisms for their role in enhancer function besides U2AF recruitment.


Subject(s)
Nuclear Proteins/metabolism , RNA Precursors/genetics , RNA Splicing , RNA, Messenger/genetics , Base Sequence , Enhancer Elements, Genetic , Exons , Humans , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Protein Structure, Tertiary , RNA Splice Sites , RNA-Binding Proteins , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors , Splicing Factor U2AF
9.
Virology ; 275(1): 145-57, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-11017796

ABSTRACT

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is required for the maintenance of the viral chromosome in latently infected, proliferating cells and plays a role in latent cycle DNA replication. EBNA-1 also functions as a positive and negative regulator of EBV gene expression. We have investigated the interaction of EBNA-1 with p32, a host mitochondrial protein that associates with EBNA-1 in EBV-positive Burkitt's lymphoma cells. Using a chromatin immunoprecipitation assay, we found that a fraction of p32 localizes to the viral latent cycle origin of DNA replication oriP in vivo. p32 binds EBNA-1 independently of other proteins or DNA. EBNA-1 variants lacking one of two p32 binding elements did not interact stably with p32 in cultured cells and were defective for both transcriptional activation of a reporter gene linked to oriP FR and replication and/or maintenance of a plasmid bearing oriP. These results support a role for p32 in transcriptional activation by EBNA-1 and suggest that p32 plays a role in EBV latent cycle DNA replication.


Subject(s)
DNA Replication/genetics , Epstein-Barr Virus Nuclear Antigens/metabolism , Hyaluronan Receptors , Proteins/metabolism , Transcriptional Activation , Virus Latency/genetics , Virus Replication/genetics , Animals , Binding Sites , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Carrier Proteins , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epstein-Barr Virus Nuclear Antigens/genetics , Gene Expression Regulation, Viral , Genes, Reporter/genetics , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/physiology , Humans , Mitochondrial Proteins , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Plasmids/genetics , Precipitin Tests , Protein Binding , Proteins/genetics , Replication Origin/genetics , Sequence Deletion/genetics , Substrate Specificity , Transfection , Tumor Cells, Cultured
10.
Mol Cell Biol ; 20(22): 8303-18, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11046128

ABSTRACT

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/metabolism , RNA Splice Sites , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Alternative Splicing , Binding Sites , Binding, Competitive , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Models, Biological , Nuclear Proteins/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoproteins/genetics , Serine-Arginine Splicing Factors
11.
J Biol Chem ; 275(37): 29170-7, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10880506

ABSTRACT

Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence. Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.


Subject(s)
Enhancer Elements, Genetic , Exons , Growth Hormone/genetics , Nuclear Proteins/metabolism , RNA Splicing , Animals , Binding Sites , Cattle , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Introns , RNA Precursors/metabolism , RNA-Binding Proteins , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors
12.
J Clin Endocrinol Metab ; 85(5): 1928-36, 2000 May.
Article in English | MEDLINE | ID: mdl-10843177

ABSTRACT

Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.


Subject(s)
Alternative Splicing , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Labor, Obstetric/metabolism , Myometrium/metabolism , Nuclear Proteins/genetics , Pregnancy/metabolism , Ribonucleoproteins/genetics , Female , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Protein Isoforms/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors , Spliceosomes/genetics
13.
J Biol Chem ; 275(33): 25411-7, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-10827081

ABSTRACT

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.


Subject(s)
Carrier Proteins , Cell Cycle Proteins/metabolism , Endoribonucleases , Intracellular Signaling Peptides and Proteins , Mitosis , Phosphoprotein Phosphatases/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cattle , Cell Cycle Proteins/chemistry , Cell Nucleus/metabolism , Cell Separation , Flow Cytometry , Fluorescent Antibody Technique , Glutathione Transferase/metabolism , HeLa Cells , Humans , Liver/metabolism , Molecular Sequence Data , Muscle, Skeletal/chemistry , Mutation , Phosphoprotein Phosphatases/chemistry , Phosphorylation , Precipitin Tests , Protein Phosphatase 1 , Protein Structure, Tertiary , Rabbits , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Schizosaccharomyces/chemistry , Schizosaccharomyces pombe Proteins , Sequence Homology, Amino Acid , Two-Hybrid System Techniques
14.
J Cell Biol ; 149(2): 307-16, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10769024

ABSTRACT

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)-rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK(3/6)-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.


Subject(s)
Alternative Splicing , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Ribonucleoproteins/metabolism , 3T3 Cells , Animals , COS Cells , Cell Line, Transformed , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , MAP Kinase Kinase 3 , MAP Kinase Kinase 6 , Mice , Osmolar Concentration , Phosphorylation , RNA-Binding Proteins/metabolism , Recombinant Proteins/biosynthesis , Signal Transduction , Simian virus 40 , Transfection , Ultraviolet Rays
15.
Mol Cell Biol ; 20(3): 1063-71, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10629063

ABSTRACT

Exonic splicing enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under splicing conditions. The selected sequences are functional and specific: they promote splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific enhancers in natural exon sequences.


Subject(s)
Enhancer Elements, Genetic , Exons , Nuclear Proteins/metabolism , RNA Splicing , Ribonucleoproteins , Animals , Base Sequence , Binding Sites , Cell Nucleus/metabolism , Cell-Free System , Consensus Sequence , HeLa Cells , Humans , Mice , Molecular Sequence Data , Promoter Regions, Genetic , RNA-Binding Proteins , Serine-Arginine Splicing Factors
18.
Proc Natl Acad Sci U S A ; 96(24): 13789-94, 1999 Nov 23.
Article in English | MEDLINE | ID: mdl-10570151

ABSTRACT

The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.


Subject(s)
Cell Cycle Proteins/metabolism , Fungal Proteins/metabolism , Protein Kinases/metabolism , Proto-Oncogene Proteins c-myb , RNA Precursors , RNA Splicing , 3T3 Cells , Animals , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , Fungal Proteins/genetics , Humans , Mice , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , RNA-Binding Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Schizosaccharomyces , Schizosaccharomyces pombe Proteins , Spliceosomes , Subcellular Fractions , Polo-Like Kinase 1
19.
Mol Biochem Parasitol ; 102(1): 103-15, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10477180

ABSTRACT

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Trans-Splicing/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan/analysis , Fluorescent Antibody Technique , Fungal Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Protozoan/metabolism , RNA, Spliced Leader/genetics , RNA-Binding Proteins/metabolism , Transcription, Genetic
20.
Proc Natl Acad Sci U S A ; 96(19): 10655-60, 1999 Sep 14.
Article in English | MEDLINE | ID: mdl-10485881

ABSTRACT

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.


Subject(s)
Catalysis , Enhancer Elements, Genetic/physiology , RNA Precursors/metabolism , RNA Splicing/physiology , Adenosine Triphosphate/pharmacology , Adenoviridae/genetics , Escherichia coli/genetics , Exons , Immunoglobulin M/metabolism , Kinetics , Models, Genetic , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotides/metabolism , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Time Factors , Trans-Splicing/physiology
SELECTION OF CITATIONS
SEARCH DETAIL
...