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. 1991 Apr;11(4):2026–2034. doi: 10.1128/mcb.11.4.2026

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

K Gröning 1, Z Palfi 1, S Gupta 1, M Cross 1, T Wolff 1, A Bindereif 1
PMCID: PMC359889  PMID: 1826044

Abstract

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

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Selected References

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

  1. Agabian N. Trans splicing of nuclear pre-mRNAs. Cell. 1990 Jun 29;61(7):1157–1160. doi: 10.1016/0092-8674(90)90674-4. [DOI] [PubMed] [Google Scholar]
  2. Bach M., Winkelmann G., Lührmann R. 20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6038–6042. doi: 10.1073/pnas.86.16.6038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banroques J., Abelson J. N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3710–3719. doi: 10.1128/mcb.9.9.3710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Bindereif A., Green M. R. Identification and functional analysis of mammalian splicing factors. Genet Eng (N Y) 1990;12:201–224. doi: 10.1007/978-1-4613-0641-2_11. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Epstein P., Reddy R., Henning D., Busch H. The nucleotide sequence of nuclear U6 (4.7 S) RNA. J Biol Chem. 1980 Sep 25;255(18):8901–8906. [PubMed] [Google Scholar]
  14. 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]
  15. Gupta S., Busch R. K., Singh R., Reddy R. Characterization of U6 small nuclear RNA cap-specific antibodies. Identification of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs. J Biol Chem. 1990 Nov 5;265(31):19137–19142. [PubMed] [Google Scholar]
  16. Gupta S., Singh R., Reddy R. Capping of U6 small nuclear RNA in vitro can be uncoupled from transcription. J Biol Chem. 1990 Jun 5;265(16):9491–9495. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Michaeli S., Roberts T. G., Watkins K. P., Agabian N. Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei. J Biol Chem. 1990 Jun 25;265(18):10582–10588. [PubMed] [Google Scholar]
  26. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  27. Mottram J., Perry K. L., Lizardi P. M., Lührmann R., Agabian N., Nelson R. G. Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs. Mol Cell Biol. 1989 Mar;9(3):1212–1223. doi: 10.1128/mcb.9.3.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murphy W. J., Watkins K. P., Agabian N. Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell. 1986 Nov 21;47(4):517–525. doi: 10.1016/0092-8674(86)90616-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Singh R., Reddy R. Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8280–8283. doi: 10.1073/pnas.86.21.8280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Sutton R. E., Boothroyd J. C. Evidence for trans splicing in trypanosomes. Cell. 1986 Nov 21;47(4):527–535. doi: 10.1016/0092-8674(86)90617-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Szkukálek A., Kiss T., Solymosy F. The 5' end of the coding region of a U6 RNA gene candidate from tomato starts with GUCC, a phylogenetically highly conserved 5' end sequence of U6 RNA. Nucleic Acids Res. 1990 Mar 11;18(5):1295–1295. doi: 10.1093/nar/18.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Zieve G. W., Sauterer R. A. Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol. 1990;25(1):1–46. doi: 10.3109/10409239009090604. [DOI] [PubMed] [Google Scholar]
  38. 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]

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