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1.
Bone ; 94: 65-74, 2017 01.
Article in English | MEDLINE | ID: mdl-27789416

ABSTRACT

BACKGROUND: Osteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit. REPORT: Whole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency. DISCUSSION: Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from 'Classical' OI. CONCLUSIONS: Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients.


Subject(s)
Mutation/genetics , Neoplasm Proteins/genetics , Osteogenesis Imperfecta/genetics , Base Sequence , Cells, Cultured , Child , Child, Preschool , Fibroblasts/pathology , Heterozygote , Humans , Infant , Infant, Newborn , Male , Neoplasm Proteins/chemistry , Osteogenesis Imperfecta/diagnostic imaging , Protein Domains , Skin/pathology , Skin/ultrastructure
2.
Biochem Soc Trans ; 33(Pt 3): 443-6, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15916537

ABSTRACT

SR proteins (serine- and arginine-rich proteins) are an evolutionarily conserved family consisting of essential pre-mRNA splicing factors. Since their discovery and initial characterization, roles of SR proteins in pre-mRNA splicing and in subsequent steps of post-transcriptional gene expression have expanded significantly. The current hypotheses suggest that SR proteins are multifunctional adaptor molecules that may couple distinct steps of RNA metabolism. In the present study, we will provide an overview of the roles of SR proteins in different steps of post-transcriptional gene expression.


Subject(s)
Arginine/metabolism , RNA Splicing , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Serine/metabolism , Humans , RNA-Binding Proteins/classification , Spliceosomes/genetics , Spliceosomes/metabolism
4.
Curr Biol ; 11(24): 1923-33, 2001 Dec 11.
Article in English | MEDLINE | ID: mdl-11747818

ABSTRACT

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.


Subject(s)
Antigens, Nuclear , Caenorhabditis elegans/genetics , Enhancer Elements, Genetic , Nuclear Matrix-Associated Proteins , Nuclear Proteins/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans/growth & development , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phenotype , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Sequence Homology, Amino Acid
5.
J Biol Chem ; 276(52): 48908-14, 2001 Dec 28.
Article in English | MEDLINE | ID: mdl-11684676

ABSTRACT

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.


Subject(s)
Alternative Splicing/genetics , Neoplasm Proteins , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , 3T3 Cells , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Cell Cycle Proteins , Cell Nucleus/metabolism , Cloning, Molecular , Genes, Reporter , HeLa Cells , Humans , Immunohistochemistry , Mice , Molecular Sequence Data , Molecular Weight , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Alignment , Serine-Arginine Splicing Factors , Tissue Distribution
6.
Hum Mol Genet ; 10(18): 1995-2011, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11555636

ABSTRACT

Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub-nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues to function for novel proteins. However, the complexity of proteins in the mammalian nucleus is too large to tackle this on a protein by protein basis. Large-scale approaches to determining protein function and sub-cellular localization are required. We have used a visual gene trap screen to identify more than 100 proteins, many of which are normal, located within compartments of the mouse nucleus. The most common discrete localizations detected are at the nucleolus and the splicing speckles and on chromosomes. Proteins at the nuclear periphery, or in other nuclear foci, have also been identified. Several of the proteins have been implicated in human disease or cancer, e.g. ATRX, HMGI-C, NBS1 and EWS, and the gene-trapped proteins provide a route into further understanding their function. We find that sequence motifs are often shared amongst proteins co-localized within the same sub-nuclear compartment. Conversely, some generally abundant motifs are lacking from the proteins concentrated in specific areas of the nucleus. This suggests that we may be able to predict sub-nuclear localization for proteins in databases based on their sequence.


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Animals , Apoptosis Regulatory Proteins , Biological Transport , Cell Cycle/physiology , Cell Differentiation/physiology , Cell Line , Cell Nucleolus/metabolism , Databases, Nucleic Acid , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryo, Nonmammalian , Gene Expression Regulation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Tumor Cells, Cultured , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
7.
Mol Cell Biol ; 21(4): 1345-59, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11158320

ABSTRACT

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.


Subject(s)
Drosophila/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Animals, Genetically Modified , Binding Sites/genetics , Cell Line , Drosophila/genetics , Female , Gene Expression , Genetic Complementation Test , HeLa Cells , Humans , Insect Proteins/genetics , Male , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphorylation , Protein Structure, Tertiary , RNA-Binding Proteins , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Amino Acid , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Sequence Homology, Amino Acid , Serine-Arginine Splicing Factors
9.
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
10.
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
11.
EMBO J ; 19(7): 1625-37, 2000 Apr 03.
Article in English | MEDLINE | ID: mdl-10747030

ABSTRACT

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.


Subject(s)
Caenorhabditis elegans/genetics , Genes, Helminth , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , DNA Primers/genetics , Helminth Proteins/genetics , Helminth Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , RNA Splicing , RNA, Helminth/genetics , RNA, Helminth/metabolism , RNA-Binding Proteins , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Sequence Homology, Amino Acid , Serine-Arginine Splicing Factors
12.
Nucleic Acids Res ; 28(24): 4822-31, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11121472

ABSTRACT

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.


Subject(s)
Alternative Splicing , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Adenovirus E1A Proteins/genetics , Amino Acid Motifs , Fibronectins/genetics , Genes, Reporter/genetics , HeLa Cells , Humans , Nuclear Proteins/genetics , Phosphoproteins/genetics , Protein Structure, Tertiary , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors , Substrate Specificity , Transfection
13.
Mol Cell ; 4(2): 251-8, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10488340

ABSTRACT

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.


Subject(s)
Alternative Splicing , Exons , Fibronectins/genetics , Promoter Regions, Genetic , RNA Polymerase II/genetics , Transcription, Genetic , Base Sequence , Enhancer Elements, Genetic , Globins/genetics , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , RNA, Messenger/genetics , Recombinant Fusion Proteins/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured , beta-Galactosidase/genetics
14.
J Cell Biol ; 143(2): 297-307, 1998 Oct 19.
Article in English | MEDLINE | ID: mdl-9786943

ABSTRACT

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.


Subject(s)
Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA Splicing/physiology , RNA-Binding Proteins/metabolism , Serine/metabolism , Transcription, Genetic/physiology , Amino Acid Sequence , Gene Deletion , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis/physiology , Nuclear Proteins/genetics , Phosphoproteins/genetics , Phosphorylation , RNA Precursors/metabolism , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors
15.
RNA ; 4(4): 430-44, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9630249

ABSTRACT

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.


Subject(s)
Alternative Splicing , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/biosynthesis , Ribonucleoproteins/biosynthesis , Adenoviridae/growth & development , Animals , Antibodies, Monoclonal , Cell Line, Transformed , Epitope Mapping , Globins/genetics , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , RNA Precursors/metabolism , RNA-Binding Proteins , Rats , Ribonucleoproteins/immunology , Serine-Arginine Splicing Factors , Spliceosomes/metabolism , Tissue Distribution
16.
Genes Dev ; 12(1): 55-66, 1998 Jan 01.
Article in English | MEDLINE | ID: mdl-9420331

ABSTRACT

The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them-SF2/ASF, SRp20, and 9G8-shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm.


Subject(s)
Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins , RNA-Binding Proteins/metabolism , 3T3 Cells , Animals , Binding Sites , Biological Transport , Cell Nucleus/metabolism , Cytoplasm/metabolism , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Mice , Nuclear Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors
17.
J Cell Biol ; 138(2): 225-38, 1997 Jul 28.
Article in English | MEDLINE | ID: mdl-9230067

ABSTRACT

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.


Subject(s)
Cell Nucleus/chemistry , Nuclear Proteins/analysis , Nuclear Proteins/genetics , RNA Splicing/genetics , Alternative Splicing/genetics , Cytoplasm/chemistry , HeLa Cells , Humans , Mutation , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins , Serine-Arginine Splicing Factors
18.
Nature ; 387(6632): 523-7, 1997 May 29.
Article in English | MEDLINE | ID: mdl-9168118

ABSTRACT

Pre-mRNA splicing is a predominantly co-transcriptional event which involves a large number of essential splicing factors. Within the mammalian cell nucleus, most splicing factors are concentrated in 20-40 distinct domains called speckles. The function of speckles and the organization of cellular transcription and pre-mRNA splicing in vivo are not well understood. We have investigated the dynamic properties of splicing factors in nuclei of living cells. Here we show that speckles are highly dynamic structures that respond specifically to activation of nearby genes. These dynamic events are dependent on RNA polymerase II transcription, and are sensitive to inhibitors of protein kinases and Ser/Thr phosphatases. When single genes are transcriptionally activated in living cells, splicing factors leave speckles in peripheral extensions and accumulate at the new sites of transcription. We conclude that one function of speckles is to supply splicing factors to active genes. Our observations demonstrate that the interphase nucleus is far more dynamic in nature than previously assumed.


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA Splicing , Cell Compartmentation , Cell Line , Green Fluorescent Proteins , Luminescent Proteins , Nuclear Proteins/genetics , Phosphorylation , RNA Polymerase II/metabolism , RNA-Binding Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine-Arginine Splicing Factors , Transcription, Genetic
19.
J Biol Chem ; 271(40): 24569-75, 1996 Oct 04.
Article in English | MEDLINE | ID: mdl-8798720

ABSTRACT

Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing, and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation. Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in vitro, but that SRPK1 has the higher specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.


Subject(s)
Alternative Splicing , Arginine/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Serine/metabolism , Amino Acid Sequence , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptide Mapping , Phosphorylation , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Substrate Specificity
20.
EMBO J ; 14(17): 4336-49, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-7556075

ABSTRACT

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.


Subject(s)
Alternative Splicing , Nuclear Proteins/biosynthesis , Nuclear Proteins/chemistry , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Amino Acid Sequence , Animals , Arginine , Base Sequence , Conserved Sequence , DNA Primers , DNA, Complementary , Drosophila , HeLa Cells , Humans , Kinetics , Mice , Molecular Sequence Data , Organ Specificity , Polymerase Chain Reaction , RNA Precursors/biosynthesis , RNA, Messenger/biosynthesis , RNA-Binding Proteins , Recombinant Proteins/biosynthesis , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Serine , Serine-Arginine Splicing Factors
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