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. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x

RNA annealing activities in HeLa nuclei.

D S Portman 1, G Dreyfuss 1
PMCID: PMC394795  PMID: 7508381

Abstract

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barnett S. F., Theiry T. A., LeStourgeon W. M. The core proteins A2 and B1 exist as (A2)3B1 tetramers in 40S nuclear ribonucleoprotein particles. Mol Cell Biol. 1991 Feb;11(2):864–871. doi: 10.1128/mcb.11.2.864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett M., Piñol-Roma S., Staknis D., Dreyfuss G., Reed R. Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro. Mol Cell Biol. 1992 Jul;12(7):3165–3175. doi: 10.1128/mcb.12.7.3165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beyer A. L., Osheim Y. N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. doi: 10.1101/gad.2.6.754. [DOI] [PubMed] [Google Scholar]
  4. Brunel F., Alzari P. M., Ferrara P., Zakin M. M. Cloning and sequencing of PYBP, a pyrimidine-rich specific single strand DNA-binding protein. Nucleic Acids Res. 1991 Oct 11;19(19):5237–5245. doi: 10.1093/nar/19.19.5237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burd C. G., Swanson M. S., Görlach M., Dreyfuss G. 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):9788–9792. doi: 10.1073/pnas.86.24.9788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buvoli M., Cobianchi F., Riva S. 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):5017–5025. doi: 10.1093/nar/20.19.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi Y. D., 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. doi: 10.1083/jcb.99.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
  9. Clouet d'Orval B., d'Aubenton Carafa Y., Sirand-Pugnet P., Gallego M., Brody E., Marie J. RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts. Science. 1991 Jun 28;252(5014):1823–1828. doi: 10.1126/science.2063195. [DOI] [PubMed] [Google Scholar]
  10. Cobianchi F., Calvio C., Stoppini M., Buvoli M., Riva S. Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):949–955. doi: 10.1093/nar/21.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cobianchi F., Karpel R. L., Williams K. R., Notario V., Wilson S. H. 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):1063–1071. [PubMed] [Google Scholar]
  12. Craig E. A. Chaperones: helpers along the pathways to protein folding. Science. 1993 Jun 25;260(5116):1902–1903. doi: 10.1126/science.8100364. [DOI] [PubMed] [Google Scholar]
  13. Dreyfuss G., Adam S. A., Choi Y. D. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription. Mol Cell Biol. 1984 Mar;4(3):415–423. doi: 10.1128/mcb.4.3.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dreyfuss G., Choi Y. D., Adam S. A. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol. 1984 Jun;4(6):1104–1114. doi: 10.1128/mcb.4.6.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  16. Eperon L. P., Graham I. R., Griffiths A. D., Eperon I. C. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988 Jul 29;54(3):393–401. doi: 10.1016/0092-8674(88)90202-4. [DOI] [PubMed] [Google Scholar]
  17. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  18. Ghetti A., Piñol-Roma S., Michael W. M., Morandi C., Dreyfuss G. hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 1992 Jul 25;20(14):3671–3678. doi: 10.1093/nar/20.14.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ghisolfi L., Joseph G., Amalric F., Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed] [Google Scholar]
  20. Gil A., Sharp P. A., Jamison S. F., Garcia-Blanco M. A. Characterization of cDNAs encoding the polypyrimidine tract-binding protein. Genes Dev. 1991 Jul;5(7):1224–1236. doi: 10.1101/gad.5.7.1224. [DOI] [PubMed] [Google Scholar]
  21. Goguel V., Rosbash M. Splice site choice and splicing efficiency are positively influenced by pre-mRNA intramolecular base pairing in yeast. Cell. 1993 Mar 26;72(6):893–901. doi: 10.1016/0092-8674(93)90578-e. [DOI] [PubMed] [Google Scholar]
  22. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  23. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  24. Görlach M., Burd C. G., Portman D. S., Dreyfuss G. The hnRNP proteins. Mol Biol Rep. 1993 Aug;18(2):73–78. doi: 10.1007/BF00986759. [DOI] [PubMed] [Google Scholar]
  25. Görlach M., Wittekind M., Beckman R. A., Mueller L., Dreyfuss G. Interaction of the RNA-binding domain of the hnRNP C proteins with RNA. EMBO J. 1992 Sep;11(9):3289–3295. doi: 10.1002/j.1460-2075.1992.tb05407.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hawkins J. D. A survey on intron and exon lengths. Nucleic Acids Res. 1988 Nov 11;16(21):9893–9908. doi: 10.1093/nar/16.21.9893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hoffman D. W., Query C. C., Golden B. L., White S. W., Keene J. D. RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2495–2499. doi: 10.1073/pnas.88.6.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Karpel R. L., Miller N. S., Fresco J. R. Mechanistic studies of ribonucleic acid renaturation by a helix-destabilizing protein. Biochemistry. 1982 Apr 27;21(9):2102–2108. doi: 10.1021/bi00538a019. [DOI] [PubMed] [Google Scholar]
  29. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  30. Konarska M. M., Padgett R. A., Sharp P. A. Trans splicing of mRNA precursors in vitro. Cell. 1985 Aug;42(1):165–171. doi: 10.1016/s0092-8674(85)80112-4. [DOI] [PubMed] [Google Scholar]
  31. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  32. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  33. Lamm G. M., Lamond A. I. Non-snRNP protein splicing factors. Biochim Biophys Acta. 1993 Jun 25;1173(3):247–265. doi: 10.1016/0167-4781(93)90122-t. [DOI] [PubMed] [Google Scholar]
  34. Lee C. G., Zamore P. D., Green M. R., Hurwitz J. RNA annealing activity is intrinsically associated with U2AF. J Biol Chem. 1993 Jun 25;268(18):13472–13478. [PubMed] [Google Scholar]
  35. Libri D., Piseri A., Fiszman M. Y. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. doi: 10.1126/science.2063196. [DOI] [PubMed] [Google Scholar]
  36. Matunis E. L., Matunis M. J., Dreyfuss G. Association of individual hnRNP proteins and snRNPs with nascent transcripts. J Cell Biol. 1993 Apr;121(2):219–228. doi: 10.1083/jcb.121.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  38. Miyatani S., Copeland N. G., Gilbert D. J., Jenkins N. A., Takeichi M. Genomic structure and chromosomal mapping of the mouse N-cadherin gene. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8443–8447. doi: 10.1073/pnas.89.18.8443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  40. Munroe S. H. Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J. 1988 Aug;7(8):2523–2532. doi: 10.1002/j.1460-2075.1988.tb03100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Munroe S. H., Dong X. F. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895–899. doi: 10.1073/pnas.89.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  43. Oberosler P., Hloch P., Ramsperger U., Stahl H. p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J. 1993 Jun;12(6):2389–2396. doi: 10.1002/j.1460-2075.1993.tb05893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Patton J. G., Mayer S. A., 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. doi: 10.1101/gad.5.7.1237. [DOI] [PubMed] [Google Scholar]
  45. Piñol-Roma S., Choi Y. D., Matunis M. J., Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. doi: 10.1101/gad.2.2.215. [DOI] [PubMed] [Google Scholar]
  46. Pontius B. W., Berg P. Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein. J Biol Chem. 1992 Jul 15;267(20):13815–13818. [PubMed] [Google Scholar]
  47. Pontius B. W., Berg P. 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):8403–8407. doi: 10.1073/pnas.87.21.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pontius B. W. Close encounters: why unstructured, polymeric domains can increase rates of specific macromolecular association. Trends Biochem Sci. 1993 May;18(5):181–186. doi: 10.1016/0968-0004(93)90111-y. [DOI] [PubMed] [Google Scholar]
  49. Racker E. Chaperones and matchmakers: inhibitors and stimulators of protein phosphorylation. Curr Top Cell Regul. 1992;33:127–143. doi: 10.1016/b978-0-12-152833-1.50013-7. [DOI] [PubMed] [Google Scholar]
  50. Sapp M., Knippers R., Richter A. DNA binding properties of a 110 kDa nucleolar protein. Nucleic Acids Res. 1986 Sep 11;14(17):6803–6820. doi: 10.1093/nar/14.17.6803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Senecoff J. F., Meagher R. B. In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs. Plant Mol Biol. 1992 Jan;18(2):219–234. doi: 10.1007/BF00034951. [DOI] [PubMed] [Google Scholar]
  52. Solnick D., Lee S. I. Amount of RNA secondary structure required to induce an alternative splice. Mol Cell Biol. 1987 Sep;7(9):3194–3198. doi: 10.1128/mcb.7.9.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Soulard M., Barque J. P., Della Valle V., Hernandez-Verdun D., Masson C., Danon F., Larsen C. J. A novel 43-kDa glycoprotein is detected in the nucleus of mammalian cells by autoantibodies from dogs with autoimmune disorders. Exp Cell Res. 1991 Mar;193(1):59–71. doi: 10.1016/0014-4827(91)90538-6. [DOI] [PubMed] [Google Scholar]
  54. Soulard M., Della Valle V., Siomi M. C., Piñol-Roma S., Codogno P., Bauvy C., Bellini M., Lacroix J. C., Monod G., Dreyfuss G. hnRNP G: sequence and characterization of a glycosylated RNA-binding protein. Nucleic Acids Res. 1993 Sep 11;21(18):4210–4217. doi: 10.1093/nar/21.18.4210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Steitz J. A. Splicing takes a holliday. Science. 1992 Aug 14;257(5072):888–889. doi: 10.1126/science.1386941. [DOI] [PubMed] [Google Scholar]
  56. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  57. Swanson M. S., 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. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Takagaki Y., MacDonald C. C., Shenk T., Manley J. L. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1403–1407. doi: 10.1073/pnas.89.4.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wittekind M., Görlach M., Friedrichs M., Dreyfuss G., Mueller L. 1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins. Biochemistry. 1992 Jul 14;31(27):6254–6265. doi: 10.1021/bi00142a013. [DOI] [PubMed] [Google Scholar]

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