• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Jul 17, 1995; 14(14): 3540–3551.
PMCID: PMC394422

The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities.

Abstract

ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.

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 (3.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Amrein H, Gorman M, Nöthiger R. The sex-determining gene tra-2 of Drosophila encodes a putative RNA binding protein. Cell. 1988 Dec 23;55(6):1025–1035. [PubMed]
  • Baker SJ, Markowitz S, Fearon ER, Willson JK, Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. [PubMed]
  • Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. [PubMed]
  • Burd CG, Dreyfuss G. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 1994 Mar 1;13(5):1197–1204. [PMC free article] [PubMed]
  • Burd CG, Matunis EL, Dreyfuss G. The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities. Mol Cell Biol. 1991 Jul;11(7):3419–3424. [PMC free article] [PubMed]
  • Cáceres JF, Krainer AR. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 1993 Dec;12(12):4715–4726. [PMC free article] [PubMed]
  • Cooper TA. In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron. J Biol Chem. 1992 Mar 15;267(8):5330–5338. [PubMed]
  • Cooper TA, Ordahl CP. Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing. Nucleic Acids Res. 1989 Oct 11;17(19):7905–7921. [PMC free article] [PubMed]
  • Crispino JD, Blencowe BJ, Sharp PA. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994 Sep 23;265(5180):1866–1869. [PubMed]
  • Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. [PMC free article] [PubMed]
  • Dirksen WP, Hampson RK, Sun Q, Rottman FM. A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA. J Biol Chem. 1994 Mar 4;269(9):6431–6436. [PubMed]
  • Fu XD. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. [PubMed]
  • Fu XD, Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. [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. [PMC free article] [PubMed]
  • Fu XD, Katz RA, Skalka AM, Maniatis T. The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA. Genes Dev. 1991 Feb;5(2):211–220. [PubMed]
  • Fu XD, Mayeda A, Maniatis T, Krainer AR. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11224–11228. [PMC free article] [PubMed]
  • Ge H, Manley JL. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. [PubMed]
  • Ge H, Zuo P, Manley JL. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. [PubMed]
  • Goralski TJ, Edström JE, Baker BS. The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins. Cell. 1989 Mar 24;56(6):1011–1018. [PubMed]
  • Green MR. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. [PubMed]
  • Hampson RK, La Follette L, Rottman FM. Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences. Mol Cell Biol. 1989 Apr;9(4):1604–1610. [PMC free article] [PubMed]
  • Harper JE, Manley JL. A novel protein factor is required for use of distal alternative 5' splice sites in vitro. Mol Cell Biol. 1991 Dec;11(12):5945–5953. [PMC free article] [PubMed]
  • Hoshijima K, Inoue K, Higuchi I, Sakamoto H, Shimura Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science. 1991 May 10;252(5007):833–836. [PubMed]
  • Huh GS, Hynes RO. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev. 1994 Jul 1;8(13):1561–1574. [PubMed]
  • Jamison SF, Crow A, Garcia-Blanco MA. The spliceosome assembly pathway in mammalian extracts. Mol Cell Biol. 1992 Oct;12(10):4279–4287. [PMC free article] [PubMed]
  • Katz RA, Skalka AM. Control of retroviral RNA splicing through maintenance of suboptimal processing signals. Mol Cell Biol. 1990 Feb;10(2):696–704. [PMC free article] [PubMed]
  • Kenan DJ, Query CC, Keene JD. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. [PubMed]
  • Kim YJ, Zuo P, Manley JL, Baker BS. The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2. Genes Dev. 1992 Dec;6(12B):2569–2579. [PubMed]
  • Kohtz JD, Jamison SF, Will CL, Zuo P, Lührmann R, Garcia-Blanco MA, Manley JL. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. [PubMed]
  • Krainer AR, Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. [PubMed]
  • Krainer AR, Conway GC, Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. [PubMed]
  • Krainer AR, Conway GC, Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. [PubMed]
  • Krainer AR, Mayeda A, Kozak D, Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. [PubMed]
  • Lavigueur A, La Branche H, Kornblihtt AR, Chabot B. 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):2405–2417. [PubMed]
  • Lee KA, Bindereif A, Green MR. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988 Mar-Apr;5(2):22–31. [PubMed]
  • Lutz CS, Alwine JC. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. [PubMed]
  • Lutz-Freyermuth C, Query CC, Keene JD. Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6393–6397. [PMC free article] [PubMed]
  • Mardon HJ, Sebastio G, Baralle FE. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Res. 1987 Oct 12;15(19):7725–7733. [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]
  • Michaud S, Reed R. A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals. Genes Dev. 1993 Jun;7(6):1008–1020. [PubMed]
  • Nietfeld W, Mentzel H, Pieler T. The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains. EMBO J. 1990 Nov;9(11):3699–3705. [PMC free article] [PubMed]
  • Reed R, Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. [PubMed]
  • Sosnowski BA, Belote JM, McKeown M. Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell. 1989 Aug 11;58(3):449–459. [PubMed]
  • Staknis D, Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. [PMC free article] [PubMed]
  • Steingrimsdottir H, Rowley G, Dorado G, Cole J, Lehmann AR. Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene. Nucleic Acids Res. 1992 Mar 25;20(6):1201–1208. [PMC free article] [PubMed]
  • Sun Q, Mayeda A, Hampson RK, Krainer AR, Rottman FM. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. [PubMed]
  • Tacke R, Goridis C. Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site. Genes Dev. 1991 Aug;5(8):1416–1429. [PubMed]
  • Tanaka K, Watakabe A, Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. [PMC free article] [PubMed]
  • Tarn WY, Steitz JA. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994 Nov 15;8(22):2704–2717. [PubMed]
  • Tian M, Maniatis T. Positive control of pre-mRNA splicing in vitro. Science. 1992 Apr 10;256(5054):237–240. [PubMed]
  • Tian M, Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. [PubMed]
  • Tian M, Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. [PubMed]
  • Tsai DE, Harper DS, Keene JD. U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. Nucleic Acids Res. 1991 Sep 25;19(18):4931–4936. [PMC free article] [PubMed]
  • Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. [PubMed]
  • Watakabe A, Tanaka K, Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. [PubMed]
  • Wu JY, Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. [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]
  • Xu R, Teng J, Cooper TA. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. [PMC free article] [PubMed]
  • Zahler AM, Lane WS, Stolk JA, Roth MB. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. [PubMed]
  • Zahler AM, Neugebauer KM, Lane WS, Roth MB. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science. 1993 Apr 9;260(5105):219–222. [PubMed]
  • Zamore PD, Green MR. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. [PMC free article] [PubMed]
  • Zamore PD, Patton JG, Green MR. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. [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]
  • Zuo P, Manley JL. Functional domains of the human splicing factor ASF/SF2. EMBO J. 1993 Dec;12(12):4727–4737. [PMC free article] [PubMed]
  • Zuo P, Manley JL. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. [PMC free article] [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links