Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI.

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  C. Burd,et al.  RNA binding specificity of hnRNP A1: significance of hnRNP A1 high‐affinity binding sites in pre‐mRNA splicing. , 1994, The EMBO journal.

[3]  A. Krainer,et al.  Functional analysis of pre‐mRNA splicing factor SF2/ASF structural domains. , 1993, The EMBO journal.

[4]  J. Manley,et al.  Functional domains of the human splicing factor ASF/SF2. , 1993, The EMBO journal.

[5]  T. Pederson,et al.  Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Tazi,et al.  Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing , 1993, Nature.

[7]  G. Dreyfuss,et al.  Association of individual hnRNP proteins and snRNPs with nascent transcripts , 1993, The Journal of cell biology.

[8]  J. G. Patton,et al.  Cloning and characterization of PSF, a novel pre-mRNA splicing factor. , 1993, Genes & development.

[9]  S. Riva,et al.  Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. , 1993, Nucleic acids research.

[10]  S. Riva,et al.  Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly. , 1992, Nucleic acids research.

[11]  R. Reed,et al.  Protein components specifically associated with prespliceosome and spliceosome complexes. , 1992, Genes & development.

[12]  G. Dreyfuss,et al.  Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro. , 1992, Molecular and cellular biology.

[13]  R. Padgett,et al.  Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing. , 1992, Nucleic acids research.

[14]  Michael R. Green,et al.  Cloning and domain structure of the mammalian splicing factor U2AF , 1992, Nature.

[15]  X. F. Dong,et al.  Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Adrian R. Krainer,et al.  Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2 , 1992, Cell.

[17]  G. Dreyfuss,et al.  Isolation of hnRNP complexes from Drosophila melanogaster , 1992, The Journal of cell biology.

[18]  G. Dreyfuss,et al.  Characterization of the major hnRNP proteins from Drosophila melanogaster , 1992, The Journal of cell biology.

[19]  M B Roth,et al.  A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription , 1991, The Journal of cell biology.

[20]  M. Frasch,et al.  Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin. , 1991, Genes & development.

[21]  F. Laski,et al.  Splicing of the Drosophila P element ORF2-ORF3 intron is inhibited in a human cell extract , 1991, Mechanisms of Development.

[22]  C. Guthrie.  Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. , 1991, Science.

[23]  J. G. Patton,et al.  Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. , 1991, Genes & development.

[24]  P. Sharp,et al.  Characterization of cDNAs encoding the polypyrimidine tract-binding protein. , 1991, Genes & development.

[25]  T. Maniatis,et al.  Mechanisms of alternative pre-mRNA splicing. , 1991, Science.

[26]  A. Kumar,et al.  Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. , 1990, Biochemistry.

[27]  P. Berg,et al.  Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[28]  B. Blencowe,et al.  Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly , 1990, Cell.

[29]  A. Krainer,et al.  Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. , 1990, Genes & development.

[30]  D. Rio,et al.  Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression. , 1990, Science.

[31]  T. Maniatis,et al.  Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus , 1990, Nature.

[32]  Barbara Neupert,et al.  A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta , 1990, Nucleic Acids Res..

[33]  P. Zamore,et al.  Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Hentze,et al.  The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[35]  D. Rio,et al.  Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Altman,et al.  Affinity chromatography with an immobilized RNA enzyme. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Halina Sierakowska,et al.  Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA , 1986, Nucleic Acids Res..

[38]  G. Dreyfuss,et al.  Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. , 1986, Science.

[39]  Y. Osheim,et al.  RNP particles at splice junction sequences on Drosophila chorion transcripts , 1985, Cell.

[40]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[41]  C. Merril,et al.  A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. , 1979, Analytical biochemistry.

[42]  I. Wool,et al.  Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5 S ribosomal ribonucleic acid. , 1979, The Journal of biological chemistry.

[43]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[44]  H. Burrell,et al.  Binding of ribosomal proteins to RNA covalently coupled to agarose. , 1977, European journal of biochemistry.

[45]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[46]  D. Rio.  Splicing of pre-mRNA: mechanism, regulation and role in development. , 1993, Current opinion in genetics & development.

[47]  P. Sharp,et al.  Splicing of precursors to mRNAs by the spliceosomes , 1993 .

[48]  C. Burd,et al.  hnRNP proteins and the biogenesis of mRNA. , 1993, Annual review of biochemistry.

[49]  M. Green,et al.  Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. , 1991, Annual review of cell biology.

[50]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[51]  E. Stellwagen.  Chromatography on immobilized reactive dyes. , 1990, Methods in enzymology.

[52]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[53]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .