Home LiteratureArticle Details
PMID: 7510636 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

The EMBO journal ·Vol. 13 ·No. 5 ·1994-03-01 ·Pages 1197-204

Burd CG, Dreyfuss G

Abstract

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.

MeSH Terms
Animals Base Sequence Binding Sites Cell Nucleus/metabolism Consensus Sequence DNA Primers Globins/genetics Heterogeneous Nuclear Ribonucleoprotein A1 Heterogeneous-Nuclear Ribonucleoprotein Group A-B Heterogeneous-Nuclear Ribonucleoproteins Introns Molecular Sequence Data Oligodeoxyribonucleotides Oligoribonucleotides/metabolism RNA/chemistry,metabolism RNA Precursors/metabolism RNA Splicing RNA-Binding Proteins/metabolism Recombinant Proteins/biosynthesis,metabolism Ribonucleoproteins/biosynthesis,metabolism Ultraviolet Rays
Chemicals
DNA Primers Heterogeneous Nuclear Ribonucleoprotein A1 Heterogeneous-Nuclear Ribonucleoprotein Group A-B Heterogeneous-Nuclear Ribonucleoproteins Oligodeoxyribonucleotides Oligoribonucleotides RNA Precursors RNA-Binding Proteins Recombinant Proteins Ribonucleoproteins RNA Globins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Burd C G
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
Dreyfuss G
References (50)
50 references, click to expand
  1. Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly.
    Proc Natl Acad Sci U S A. 1990 Nov;87(21):8403-7 PMID: 2236048
  2. Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain.
    J Biol Chem. 1990 Oct 5;265(28):17094-100 PMID: 2145269
  3. Characterization of the major hnRNP proteins from Drosophila melanogaster.
    J Cell Biol. 1992 Jan;116(2):257-69 PMID: 1730754
  4. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2.
    Cell. 1992 Jan 24;68(2):365-75 PMID: 1531115
  5. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing.
    Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895-9 PMID: 1371011
  6. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm.
    Nature. 1992 Feb 20;355(6362):730-2 PMID: 1371331
  7. Cloning and domain structure of the mammalian splicing factor U2AF.
    Nature. 1992 Feb 13;355(6361):609-14 PMID: 1538748
  8. Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro.
    Mol Cell Biol. 1992 Jul;12(7):3165-75 PMID: 1620124
  9. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.
    EMBO J. 1992 Jul;11(7):2655-64 PMID: 1628625
  10. Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.
    EMBO J. 1992 Sep;11(9):3289-95 PMID: 1380452
  11. Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth.
    Mol Cell Biol. 1992 Oct;12(10):4449-55 PMID: 1406633
  12. 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.
    Nucleic Acids Res. 1992 Oct 11;20(19):5017-25 PMID: 1329035
  13. The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes.
    Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10969-73 PMID: 1332064
  14. Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.
    J Mol Biol. 1992 Dec 5;228(3):862-9 PMID: 1469719
  15. hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn.
    Nucleic Acids Res. 1992 Dec 25;20(24):6461-4 PMID: 1282701
  16. Selective enrichment of RNA species for tight binding to Escherichia coli rho factor.
    FASEB J. 1993 Jan;7(1):201-7 PMID: 7678562
  17. Mammalian heterogeneous ribonucleoprotein A1 and its constituent domains. Nucleic acid interaction, structural stability and self-association.
    J Mol Biol. 1993 Feb 20;229(4):873-89 PMID: 8445653
  18. Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.
    Nucleic Acids Res. 1993 Feb 25;21(4):949-55 PMID: 8451194
  19. Association of individual hnRNP proteins and snRNPs with nascent transcripts.
    J Cell Biol. 1993 Apr;121(2):219-28 PMID: 8468343
  20. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.
    Mol Cell Biol. 1993 May;13(5):2993-3001 PMID: 8474457
  21. Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.
    Mol Cell Biol. 1993 Jul;13(7):4301-10 PMID: 8321232
  22. hnRNP proteins and the biogenesis of mRNA.
    Annu Rev Biochem. 1993;62:289-321 PMID: 8352591
  23. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7642-6 PMID: 8395052
  24. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.
    EMBO J. 1993 Sep;12(9):3607-17 PMID: 8253084
  25. RNA annealing activities in HeLa nuclei.
    EMBO J. 1994 Jan 1;13(1):213-21 PMID: 7508381
  26. Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions.
    Biochemistry. 1991 Mar 19;30(11):2968-76 PMID: 1848781
  27. The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.
    Mol Cell Biol. 1991 Jul;11(7):3419-24 PMID: 1675426
  28. Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.
    J Mol Biol. 1991 Sep 20;221(2):693-709 PMID: 1656054
  29. 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):4931-6 PMID: 1717938
  30. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA.
    Cell. 1991 Nov 1;67(3):529-36 PMID: 1934059
  31. SELEXION. Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis.
    J Mol Biol. 1991 Dec 5;222(3):739-61 PMID: 1721092
  32. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1.
    Biochemistry. 1990 Dec 4;29(48):10717-22 PMID: 1703006
  33. A catalogue of splice junction sequences.
    Nucleic Acids Res. 1982 Jan 22;10(2):459-72 PMID: 7063411
  34. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site.
    Biochemistry. 1983 May 24;22(11):2601-10 PMID: 6347247
  35. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate.
    Cell. 1984 Jun;37(2):415-27 PMID: 6722880
  36. Structure of rodent helix-destabilizing protein revealed by cDNA cloning.
    J Biol Chem. 1986 Mar 15;261(8):3536-43 PMID: 3005291
  37. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing.
    Science. 1986 Mar 28;231(4745):1534-9 PMID: 3952495
  38. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles.
    Annu Rev Cell Biol. 1986;2:459-98 PMID: 3548774
  39. Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences.
    J Mol Biol. 1987 May 20;195(2):247-59 PMID: 3656413
  40. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.
    Nucleic Acids Res. 1987 Nov 11;15(21):8783-98 PMID: 3684574
  41. Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids.
    J Biol Chem. 1988 Jan 15;263(2):1063-71 PMID: 2447078
  42. Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein.
    J Biol Chem. 1988 Mar 5;263(7):3307-13 PMID: 2830282
  43. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.
    Mol Cell Biol. 1988 May;8(5):2237-41 PMID: 3386636
  44. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns.
    EMBO J. 1988 Nov;7(11):3519-29 PMID: 3208740
  45. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation.
    Trends Biochem Sci. 1988 Mar;13(3):86-91 PMID: 3072706
  46. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts.
    Proc Natl Acad Sci U S A. 1989 Dec;86(24):9788-92 PMID: 2557628
  47. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.
    Science. 1990 Aug 3;249(4968):505-10 PMID: 2200121
  48. 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):6393-7 PMID: 1696729
  49. The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.
    EMBO J. 1990 Nov;9(11):3699-705 PMID: 2209558
  50. 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):6595-600 PMID: 2251120
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1994-03-01
Pages
1197-204
Language
English
Region
England
NLM ID
8208664
PMCID
PMC394929
Subset
IM
External Links
PubMed source PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2