PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1992 July; 12(7): 3165–3175.
PMCID: PMC364531

Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro.

Abstract

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abmayr SM, Reed R, Maniatis T. Identification of a functional mammalian spliceosome containing unspliced pre-mRNA. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7216–7220. [PubMed]
  • Beyer AL, Osheim YN. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. [PubMed]
  • Bernstein SI, Hansen CJ, Becker KD, Wassenberg DR, 2nd, Roche ES, Donady JJ, Emerson CP., Jr Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain. Mol Cell Biol. 1986 Jul;6(7):2511–2519. [PMC free article] [PubMed]
  • Bindereif A, Green MR. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. [PubMed]
  • Brody E, Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. [PubMed]
  • Buvoli M, Cobianchi F, Biamonti G, Riva S. Recombinant hnRNP protein A1 and its N-terminal domain show preferential affinity for oligodeoxynucleotides homologous to intron/exon acceptor sites. Nucleic Acids Res. 1990 Nov 25;18(22):6595–6600. [PMC free article] [PubMed]
  • Choi YD, Dreyfuss G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7471–7475. [PubMed]
  • Choi YD, Dreyfuss G. Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells. J Cell Biol. 1984 Dec;99(6):1997–1204. [PMC free article] [PubMed]
  • Choi YD, Grabowski PJ, Sharp PA, Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. [PubMed]
  • Chung SY, Wooley J. Set of novel, conserved proteins fold pre-messenger RNA into ribonucleosomes. Proteins. 1986 Nov;1(3):195–210. [PubMed]
  • Conway G, Wooley J, Bibring T, LeStourgeon WM. Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles. Mol Cell Biol. 1988 Jul;8(7):2884–2895. [PMC free article] [PubMed]
  • Dreyfuss G. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol. 1986;2:459–498. [PubMed]
  • Dreyfuss G, Adam SA, Choi YD. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription. Mol Cell Biol. 1984 Mar;4(3):415–423. [PMC free article] [PubMed]
  • Frendewey D, Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. [PubMed]
  • Fu XD, Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. [PubMed]
  • García-Blanco MA, Jamison SF, Sharp PA. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. [PubMed]
  • Gil A, Sharp PA, Jamison SF, Garcia-Blanco MA. Characterization of cDNAs encoding the polypyrimidine tract-binding protein. Genes Dev. 1991 Jul;5(7):1224–1236. [PubMed]
  • Grabowski PJ, Seiler SR, Sharp PA. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. [PubMed]
  • Grabowski PJ, Sharp PA. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 1986 Sep 19;233(4770):1294–1299. [PubMed]
  • Green MR. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. [PubMed]
  • Hartley SE, Callan HG. RNA transcription on the giant lateral loops of the lampbrush chromosomes of the American newt Notophthalmus viridescens. J Cell Sci. 1978 Dec;34:279–288. [PubMed]
  • Konarska MM, Sharp PA. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. [PubMed]
  • Konarska MM, Sharp PA. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. [PubMed]
  • Krainer AR, Maniatis T, Ruskin B, Green MR. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. [PubMed]
  • Krämer A. Presplicing complex formation requires two proteins and U2 snRNP. Genes Dev. 1988 Sep;2(9):1155–1167. [PubMed]
  • Krämer A, Utans U. Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs. EMBO J. 1991 Jun;10(6):1503–1509. [PubMed]
  • Mattaj IW. Splicing stories and poly(A) tales: an update on RNA processing and transport. Curr Opin Cell Biol. 1990 Jun;2(3):528–538. [PubMed]
  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. [PMC free article] [PubMed]
  • Michaud S, Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991 Dec;5(12B):2534–2546. [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Osheim YN, Miller OL, Jr, Beyer AL. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell. 1985 Nov;43(1):143–151. [PubMed]
  • Pandolfo M, Valentini O, Biamonti G, Rossi P, Riva S. Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose. Eur J Biochem. 1987 Jan 2;162(1):213–220. [PubMed]
  • Parker R, Siliciano PG, Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. [PubMed]
  • Patton JG, Mayer SA, Tempst P, Nadal-Ginard B. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 1991 Jul;5(7):1237–1251. [PubMed]
  • Piñol-Roma S, Choi YD, Matunis MJ, Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. [PubMed]
  • Piñol-Roma S, Swanson MS, Gall JG, Dreyfuss G. A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts. J Cell Biol. 1989 Dec;109(6 Pt 1):2575–2587. [PMC free article] [PubMed]
  • Reed R. Protein composition of mammalian spliceosomes assembled in vitro. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8031–8035. [PubMed]
  • Reed R, Griffith J, Maniatis T. Purification and visualization of native spliceosomes. Cell. 1988 Jun 17;53(6):949–961. [PubMed]
  • Reed R, Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. [PubMed]
  • Séraphin B, Kretzner L, Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. [PubMed]
  • Sierakowska H, Szer W, Furdon PJ, Kole R. Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA. Nucleic Acids Res. 1986 Jul 11;14(13):5241–5254. [PMC free article] [PubMed]
  • Siliciano PG, Guthrie C. 5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 1988 Oct;2(10):1258–1267. [PubMed]
  • Smith CW, Porro EB, Patton JG, Nadal-Ginard B. Scanning from an independently specified branch point defines the 3' splice site of mammalian introns. Nature. 1989 Nov 16;342(6247):243–247. [PubMed]
  • Swanson MS, Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. [PubMed]
  • Swanson MS, Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. [PMC free article] [PubMed]
  • Wu J, Manley JL. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. [PubMed]
  • Zamore PD, Green MR. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991 Jan;10(1):207–214. [PubMed]
  • Zhuang Y, Weiner AM. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. [PubMed]
  • Zhuang Y, Weiner AM. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)