Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis (original) (raw)

Abstract

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.

345

Images in this article

Selected References

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

  1. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  2. Barabino S. M., Sproat B. S., Ryder U., Blencowe B. J., Lamond A. I. Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA. EMBO J. 1989 Dec 20;8(13):4171–4178. doi: 10.1002/j.1460-2075.1989.tb08602.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bindereif A., Wolff T., Green M. R. Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes. EMBO J. 1990 Jan;9(1):251–255. doi: 10.1002/j.1460-2075.1990.tb08102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Black D. L., Pinto A. L. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. Mol Cell Biol. 1989 Aug;9(8):3350–3359. doi: 10.1128/mcb.9.8.3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Black D. L., Steitz J. A. Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein. Cell. 1986 Aug 29;46(5):697–704. doi: 10.1016/0092-8674(86)90345-4. [DOI] [PubMed] [Google Scholar]
  7. Blencowe B. J., Sproat B. S., Ryder U., Barabino S., Lamond A. I. Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides. Cell. 1989 Nov 3;59(3):531–539. doi: 10.1016/0092-8674(89)90036-6. [DOI] [PubMed] [Google Scholar]
  8. Bringmann P., Appel B., Rinke J., Reuter R., Theissen H., Lührmann R. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J. 1984 Jun;3(6):1357–1363. doi: 10.1002/j.1460-2075.1984.tb01977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
  10. Brow D. A., Guthrie C. Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 1990 Aug;4(8):1345–1356. doi: 10.1101/gad.4.8.1345. [DOI] [PubMed] [Google Scholar]
  11. Burgess S., Couto J. R., Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. doi: 10.1016/0092-8674(90)90086-t. [DOI] [PubMed] [Google Scholar]
  12. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  13. Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. doi: 10.1038/349487a0. [DOI] [PubMed] [Google Scholar]
  14. Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Datta B., Weiner A. M. Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing. Nature. 1991 Aug 29;352(6338):821–824. doi: 10.1038/352821a0. [DOI] [PubMed] [Google Scholar]
  16. Dignam J. D., Lebovitz R. M., Roeder R. G. 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. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fabrizio P., Abelson J. Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing. Science. 1990 Oct 19;250(4979):404–409. doi: 10.1126/science.2145630. [DOI] [PubMed] [Google Scholar]
  18. Fabrizio P., McPheeters D. S., Abelson J. In vitro assembly of yeast U6 snRNP: a functional assay. Genes Dev. 1989 Dec;3(12B):2137–2150. doi: 10.1101/gad.3.12b.2137. [DOI] [PubMed] [Google Scholar]
  19. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  20. Gröning K., Palfi Z., Gupta S., Cross M., Wolff T., Bindereif A. A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems. Mol Cell Biol. 1991 Apr;11(4):2026–2034. doi: 10.1128/mcb.11.4.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
  22. Habets W. J., Hoet M. H., De Jong B. A., Van der Kemp A., Van Venrooij W. J. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies. J Immunol. 1989 Oct 15;143(8):2560–2566. [PubMed] [Google Scholar]
  23. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  24. Hamm J., Mattaj I. W. An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis. EMBO J. 1989 Dec 20;8(13):4179–4187. doi: 10.1002/j.1460-2075.1989.tb08603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hashimoto C., Steitz J. A. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res. 1984 Apr 11;12(7):3283–3293. doi: 10.1093/nar/12.7.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hausner T. P., Giglio L. M., Weiner A. M. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 1990 Dec;4(12A):2146–2156. doi: 10.1101/gad.4.12a.2146. [DOI] [PubMed] [Google Scholar]
  27. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Inoue H., Hayase Y., Iwai S., Ohtsuka E. Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 1987 May 11;215(2):327–330. doi: 10.1016/0014-5793(87)80171-0. [DOI] [PubMed] [Google Scholar]
  29. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  30. Lamm G. M., Blencowe B. J., Sproat B. S., Iribarren A. M., Ryder U., Lamond A. I. Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts. Nucleic Acids Res. 1991 Jun 25;19(12):3193–3198. doi: 10.1093/nar/19.12.3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamond A. I., Konarska M. M., Grabowski P. J., Sharp P. A. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc Natl Acad Sci U S A. 1988 Jan;85(2):411–415. doi: 10.1073/pnas.85.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  33. Madhani H. D., Bordonné R., Guthrie C. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 1990 Dec;4(12B):2264–2277. doi: 10.1101/gad.4.12b.2264. [DOI] [PubMed] [Google Scholar]
  34. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  35. Mattaj I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. doi: 10.1016/0092-8674(86)90072-3. [DOI] [PubMed] [Google Scholar]
  36. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  37. McPheeters D. S., Fabrizio P., Abelson J. In vitro reconstitution of functional yeast U2 snRNPs. Genes Dev. 1989 Dec;3(12B):2124–2136. doi: 10.1101/gad.3.12b.2124. [DOI] [PubMed] [Google Scholar]
  38. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  39. Okano Y., Medsger T. A., Jr Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis. J Immunol. 1991 Jan 15;146(2):535–542. [PubMed] [Google Scholar]
  40. Palfi Z., Günzl A., Cross M., Bindereif A. Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9097–9101. doi: 10.1073/pnas.88.20.9097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pikielny C. W., Bindereif A., Green M. R. In vitro reconstitution of snRNPs: a reconstituted U4/U6 snRNP participates in splicing complex formation. Genes Dev. 1989 Apr;3(4):479–487. doi: 10.1101/gad.3.4.479. [DOI] [PubMed] [Google Scholar]
  42. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  43. Reich C., Wise J. A. Evolutionary origin of the U6 small nuclear RNA intron. Mol Cell Biol. 1990 Oct;10(10):5548–5552. doi: 10.1128/mcb.10.10.5548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rinke J., Appel B., Digweed M., Lührmann R. Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking. J Mol Biol. 1985 Oct 20;185(4):721–731. doi: 10.1016/0022-2836(85)90057-9. [DOI] [PubMed] [Google Scholar]
  45. Roiha H., Shuster E. O., Brow D. A., Guthrie C. Small nuclear RNAs from budding yeasts: phylogenetic comparisons reveal extensive size variation. Gene. 1989 Oct 15;82(1):137–144. doi: 10.1016/0378-1119(89)90038-3. [DOI] [PubMed] [Google Scholar]
  46. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  47. Shuster E. O., Guthrie C. Human U2 snRNA can function in pre-mRNA splicing in yeast. Nature. 1990 May 17;345(6272):270–273. doi: 10.1038/345270a0. [DOI] [PubMed] [Google Scholar]
  48. Singh R., Gupta S., Reddy R. Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA. Mol Cell Biol. 1990 Mar;10(3):939–946. doi: 10.1128/mcb.10.3.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sproat B. S., Lamond A. I., Beijer B., Neuner P., Ryder U. Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases. Nucleic Acids Res. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Strauss E. J., Guthrie C. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 1991 Apr;5(4):629–641. doi: 10.1101/gad.5.4.629. [DOI] [PubMed] [Google Scholar]
  51. Séraphin B., Abovich N., Rosbash M. Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly. Nucleic Acids Res. 1991 Jul 25;19(14):3857–3860. doi: 10.1093/nar/19.14.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tani T., Ohshima Y. The gene for the U6 small nuclear RNA in fission yeast has an intron. Nature. 1989 Jan 5;337(6202):87–90. doi: 10.1038/337087a0. [DOI] [PubMed] [Google Scholar]
  53. Tani T., Ohshima Y. mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing. Genes Dev. 1991 Jun;5(6):1022–1031. doi: 10.1101/gad.5.6.1022. [DOI] [PubMed] [Google Scholar]
  54. Vankan P., McGuigan C., Mattaj I. W. Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes. EMBO J. 1990 Oct;9(10):3397–3404. doi: 10.1002/j.1460-2075.1990.tb07541.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wersig C., Bindereif A. Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly. Nucleic Acids Res. 1990 Nov 11;18(21):6223–6229. doi: 10.1093/nar/18.21.6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wu J. A., Manley J. L. Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. Nature. 1991 Aug 29;352(6338):818–821. doi: 10.1038/352818a0. [DOI] [PubMed] [Google Scholar]
  57. Zillmann M., Zapp M. L., Berget S. M. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol Cell Biol. 1988 Feb;8(2):814–821. doi: 10.1128/mcb.8.2.814. [DOI] [PMC free article] [PubMed] [Google Scholar]