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PMID: 10072387 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene.

Genes & development ·Vol. 13 ·No. 5 ·1999-03-01 ·Pages 593-606

Chen CD, Kobayashi R, Helfman DM

Abstract

In the rat beta-tropomyosin (beta-TM) gene, exons 6 and 7 are spliced alternatively in a mutually exclusive manner. Exon 6 is included in mRNA encoding nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle beta-TM. Previously, we demonstrated that a six nucleotide mutation at the 5' end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupting a putative secondary structure. The sequence in exon 7 acts as a bona fide exonic splicing silencer (ESS), which is bound specifically by a trans-acting factor. Isolation and peptide sequencing reveal that this factor is hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Binding of hnRNP H correlates with the ESS activity. Furthermore, addition of antibodies that specifically recognizes hnRNP H to the splicing reactions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP H. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue- and developmental-specific alternative splicing.

MeSH Terms
Alternative Splicing Animals Base Sequence Exons Gene Expression Regulation HeLa Cells Heterogeneous-Nuclear Ribonucleoprotein Group F-H Heterogeneous-Nuclear Ribonucleoproteins Humans Molecular Sequence Data Mutagenesis RNA Rats Recombinant Proteins/metabolism Ribonucleoproteins/metabolism Tropomyosin/genetics
Chemicals
Heterogeneous-Nuclear Ribonucleoprotein Group F-H Heterogeneous-Nuclear Ribonucleoproteins Recombinant Proteins Ribonucleoproteins Tropomyosin RNA
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Chen C D
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Kobayashi R
Helfman D M
References (66)
66 references, click to expand
  1. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.
    RNA. 1998 Jan;4(1):85-100 PMID: 9436911
  2. Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.
    EMBO J. 1998 May 15;17(10):2904-13 PMID: 9582284
  3. Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation.
    Genes Dev. 1991 Nov;5(11):2071-85 PMID: 1936994
  4. Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells.
    Genes Dev. 1991 Nov;5(11):2096-107 PMID: 1936995
  5. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
    Nucleic Acids Res. 1983 Mar 11;11(5):1475-89 PMID: 6828386
  6. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.
    Cell. 1984 Apr;36(4):993-1005 PMID: 6323033
  7. Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro.
    Cell. 1987 Jul 3;50(1):31-9 PMID: 2954651
  8. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene.
    Cell. 1987 Aug 28;50(5):739-47 PMID: 2441872
  9. Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo.
    Genes Dev. 1988 Dec;2(12A):1627-38 PMID: 3215513
  10. Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.
    Mol Cell Biol. 1989 Apr;9(4):1604-10 PMID: 2725519
  11. Branch point selection in alternative splicing of tropomyosin pre-mRNAs.
    Nucleic Acids Res. 1989 Jul 25;17(14):5633-50 PMID: 2762151
  12. The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points.
    RNA. 1998 Jun;4(6):613-25 PMID: 9622121
  13. Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA.
    RNA. 1999 Mar;5(3):378-94 PMID: 10094307
  14. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure.
    Science. 1991 Jun 28;252(5014):1842-5 PMID: 2063196
  15. Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.
    Genes Dev. 1990 Jan;4(1):98-110 PMID: 2307372
  16. Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript.
    Nature. 1990 Mar 29;344(6265):461-3 PMID: 1690860
  17. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro.
    Cell. 1990 Jul 13;62(1):25-34 PMID: 2163768
  18. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites.
    Cell. 1990 Jul 13;62(1):35-42 PMID: 2364434
  19. Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene.
    Mol Cell Biol. 1990 Oct;10(10):5036-46 PMID: 2398885
  20. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila.
    Science. 1991 May 10;252(5007):833-6 PMID: 1902987
  21. Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro.
    Cell. 1991 May 17;65(4):579-86 PMID: 1674449
  22. RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts.
    Science. 1991 Jun 28;252(5014):1823-8 PMID: 2063195
  23. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2.
    Cell. 1992 Jan 24;68(2):365-75 PMID: 1531115
  24. SR proteins: a conserved family of pre-mRNA splicing factors.
    Genes Dev. 1992 May;6(5):837-47 PMID: 1577277
  25. Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro.
    Cell. 1992 May 29;69(5):795-807 PMID: 1591778
  26. Sex differentiation: the role of alternative splicing.
    Curr Opin Genet Dev. 1992 Apr;2(2):299-303 PMID: 1638125
  27. Binding of the Drosophila transformer and transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing.
    Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8092-6 PMID: 1518835
  28. Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.
    J Biol Chem. 1992 Dec 15;267(35):25480-7 PMID: 1460042
  29. The role of exon sequences in splice site selection.
    Genes Dev. 1993 Mar;7(3):407-18 PMID: 8449402
  30. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA.
    Nature. 1993 Mar 11;362(6416):171-5 PMID: 7680770
  31. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.
    Mol Cell Biol. 1993 Jun;13(6):3660-74 PMID: 8388541
  32. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA.
    Cell. 1993 Jul 16;74(1):105-14 PMID: 8334698
  33. hnRNP proteins and the biogenesis of mRNA.
    Annu Rev Biochem. 1993;62:289-321 PMID: 8352591
  34. Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7764-8 PMID: 8356082
  35. cis-elements involved in alternative splicing in the rat beta-tropomyosin gene: the 3'-splice site of the skeletal muscle exon 7 is the major site of blockage in nonmuscle cells.
    Nucleic Acids Res. 1993 Oct 11;21(20):4762-8 PMID: 8233825
  36. Splicing of pre-mRNA: mechanism, regulation and role in development.
    Curr Opin Genet Dev. 1993 Aug;3(4):574-84 PMID: 8241769
  37. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding.
    Genes Dev. 1993 Dec;7(12A):2405-17 PMID: 8253386
  38. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.
    Cell. 1993 Dec 17;75(6):1061-70 PMID: 8261509
  39. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer.
    Genes Dev. 1993 Dec;7(12B):2598-608 PMID: 8276242
  40. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.
    Nucleic Acids Res. 1994 Mar 25;22(6):1018-22 PMID: 8152907
  41. The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization.
    Nucleic Acids Res. 1994 Mar 25;22(6):1059-67 PMID: 7512260
  42. Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.
    Nucleic Acids Res. 1994 Jun 25;22(12):2318-25 PMID: 8036160
  43. The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo.
    Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6924-8 PMID: 8041722
  44. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.
    Science. 1994 Sep 16;265(5179):1706-9 PMID: 8085156
  45. Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding.
    Nucleic Acids Res. 1994 Sep 25;22(19):3854-60 PMID: 7937104
  46. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element.
    Genes Dev. 1994 Jul 1;8(13):1561-74 PMID: 7958840
  47. Synergistic interactions between two distinct elements of a regulated splicing enhancer.
    Genes Dev. 1995 Feb 1;9(3):284-93 PMID: 7867927
  48. Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.
    Mol Cell Biol. 1995 Aug;15(8):4597-605 PMID: 7623851
  49. Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.
    Mol Cell Biol. 1995 Aug;15(8):4606-15 PMID: 7623852
  50. Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.
    Mol Cell Biol. 1995 Sep;15(9):4825-34 PMID: 7651400
  51. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.
    Mol Cell Biol. 1995 Sep;15(9):4898-907 PMID: 7651409
  52. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.
    Nucleic Acids Res. 1995 Sep 11;23(17):3501-7 PMID: 7567462
  53. The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event.
    Genes Dev. 1995 Nov 1;9(21):2659-71 PMID: 7590243
  54. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.
    Mol Cell Biol. 1995 Dec;15(12):7135-42 PMID: 8524281
  55. Regulation of alternative 3' splice site selection by constitutive splicing factors.
    RNA. 1995 May;1(3):234-45 PMID: 7489496
  56. Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes.
    J Biol Chem. 1995 Dec 1;270(48):28780-9 PMID: 7499401
  57. An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.
    RNA. 1996 Apr;2(4):342-53 PMID: 8634915
  58. Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA.
    Nature. 1996 Jun 6;381(6582):535-8 PMID: 8632829
  59. The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing.
    Genes Dev. 1996 Jun 1;10(11):1356-68 PMID: 8647433
  60. Selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor.
    J Virol. 1996 Jul;70(7):4691-9 PMID: 8676495
  61. Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.
    Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8425-30 PMID: 8710887
  62. Biochemistry and regulation of pre-mRNA splicing.
    Curr Opin Cell Biol. 1996 Jun;8(3):331-9 PMID: 8743883
  63. A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.
    Genes Dev. 1997 Apr 15;11(8):1023-36 PMID: 9136930
  64. Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo.
    RNA. 1997 Jul;3(7):764-78 PMID: 9214659
  65. The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.
    Mol Cell Biol. 1997 Aug;17(8):4667-76 PMID: 9234723
  66. A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart.
    RNA. 1997 Sep;3(9):996-1015 PMID: 9292499
Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.13.5.593
Published
1999-03-01
Pages
593-606
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC316507
Subset
IM
Grants
NCI NIH HHS · CA95508 · United States
NIGMS NIH HHS · GM43049 · United States
External Links
PubMed source DOI: 10.1101/gad.13.5.593 PMC Full Text View journal details
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