Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Apr;11(4):1531–1542. doi: 10.1002/j.1460-2075.1992.tb05198.x

Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.

M Beltrame 1, D Tollervey 1
PMCID: PMC556602  PMID: 1563354

Abstract

It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.

Full text

PDF
1531

Images in this article

1532Image
on p.1532
1533Image
on p.1533
1534Image
on p.1534
1535Image
on p.1535
1536Image
on p.1536
1537Image
on p.1537
1538Image
on p.1538
1539Image
on p.1539
1539Image
on p.1539
1540Image
on p.1540

Selected References

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

  1. Bachellerie J. P., Michot B., Raynal F. Recognition signals for mouse pre-rRNA processing. A potential role for U3 nucleolar RNA. Mol Biol Rep. 1983 May;9(1-2):79–86. doi: 10.1007/BF00777477. [DOI] [PubMed] [Google Scholar]
  2. Boorstein W. R., Craig E. A. Primer extension analysis of RNA. Methods Enzymol. 1989;180:347–369. doi: 10.1016/0076-6879(89)80111-9. [DOI] [PubMed] [Google Scholar]
  3. Clark M. W., Yip M. L., Campbell J., Abelson J. SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs. J Cell Biol. 1990 Nov;111(5 Pt 1):1741–1751. doi: 10.1083/jcb.111.5.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crouch R. J., Kanaya S., Earl P. L. A model for the involvement of the small nucleolar RNA (U3) in processing eukaryotic ribosomal RNA. Mol Biol Rep. 1983 May;9(1-2):75–78. doi: 10.1007/BF00777476. [DOI] [PubMed] [Google Scholar]
  5. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hughes J. M., Ares M., Jr Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 1991 Dec;10(13):4231–4239. doi: 10.1002/j.1460-2075.1991.tb05001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hughes J. M., Konings D. A., Cesareni G. The yeast homologue of U3 snRNA. EMBO J. 1987 Jul;6(7):2145–2155. doi: 10.1002/j.1460-2075.1987.tb02482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jansen R. P., Hurt E. C., Kern H., Lehtonen H., Carmo-Fonseca M., Lapeyre B., Tollervey D. Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast. J Cell Biol. 1991 May;113(4):715–729. doi: 10.1083/jcb.113.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kass S., Craig N., Sollner-Webb B. Primary processing of mammalian rRNA involves two adjacent cleavages and is not species specific. Mol Cell Biol. 1987 Aug;7(8):2891–2898. doi: 10.1128/mcb.7.8.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kass S., Sollner-Webb B. The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking. Mol Cell Biol. 1990 Sep;10(9):4920–4931. doi: 10.1128/mcb.10.9.4920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell. 1990 Mar 23;60(6):897–908. doi: 10.1016/0092-8674(90)90338-f. [DOI] [PubMed] [Google Scholar]
  13. Klootwijk J., Planta R. J. Isolation and characterization of yeast ribosomal RNA precursors and preribosomes. Methods Enzymol. 1989;180:96–109. doi: 10.1016/0076-6879(89)80095-3. [DOI] [PubMed] [Google Scholar]
  14. Li H. D., Zagorski J., Fournier M. J. Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Mar;10(3):1145–1152. doi: 10.1128/mcb.10.3.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maden B. E., Moss M., Salim M. Nucleotide sequence of an external transcribed spacer in Xenopus laevis rDNA: sequences flanking the 5' and 3' ends of 18S rRNA are non-complementary. Nucleic Acids Res. 1982 Apr 10;10(7):2387–2398. doi: 10.1093/nar/10.7.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maser R. L., Calvet J. P. U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6523–6527. doi: 10.1073/pnas.86.17.6523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Michot B., Bachellerie J. P. Secondary structure of the 5' external transcribed spacer of vertebrate pre-rRNA. Presence of phylogenetically conserved features. Eur J Biochem. 1991 Feb 14;195(3):601–609. doi: 10.1111/j.1432-1033.1991.tb15743.x. [DOI] [PubMed] [Google Scholar]
  18. Musters W., Boon K., van der Sande C. A., van Heerikhuizen H., Planta R. J. Functional analysis of transcribed spacers of yeast ribosomal DNA. EMBO J. 1990 Dec;9(12):3989–3996. doi: 10.1002/j.1460-2075.1990.tb07620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Musters W., Venema J., van der Linden G., van Heerikhuizen H., Klootwijk J., Planta R. J. A system for the analysis of yeast ribosomal DNA mutations. Mol Cell Biol. 1989 Feb;9(2):551–559. doi: 10.1128/mcb.9.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myslinski E., Ségault V., Branlant C. An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae. Science. 1990 Mar 9;247(4947):1213–1216. doi: 10.1126/science.1690452. [DOI] [PubMed] [Google Scholar]
  21. Parker K. A., Bruzik J. P., Steitz J. A. An in vitro interaction between the human U3 snRNP and 28S rRNA sequences near the alpha-sarcin site. Nucleic Acids Res. 1988 Nov 25;16(22):10493–10509. doi: 10.1093/nar/16.22.10493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Parker K. A., Steitz J. A. Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol Cell Biol. 1987 Aug;7(8):2899–2913. doi: 10.1128/mcb.7.8.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Porter G. L., Brennwald P. J., Holm K. A., Wise J. A. The sequence of U3 from Schizosaccharomyces pombe suggests structural divergence of this snRNA between metazoans and unicellular eukaryotes. Nucleic Acids Res. 1988 Nov 11;16(21):10131–10152. doi: 10.1093/nar/16.21.10131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prestayko A. W., Tonato M., Busch H. Low molecular weight RNA associated with 28 s nucleolar RNA. J Mol Biol. 1970 Feb 14;47(3):505–515. doi: 10.1016/0022-2836(70)90318-9. [DOI] [PubMed] [Google Scholar]
  25. Savino R., Gerbi S. A. In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. EMBO J. 1990 Jul;9(7):2299–2308. doi: 10.1002/j.1460-2075.1990.tb07401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stroke I. L., Weiner A. M. The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors. J Mol Biol. 1989 Dec 5;210(3):497–512. doi: 10.1016/0022-2836(89)90126-5. [DOI] [PubMed] [Google Scholar]
  28. Tague B. W., Gerbi S. A. Processing of the large rRNA precursor: two proposed categories of RNA-RNA interactions in eukaryotes. J Mol Evol. 1984;20(3-4):362–367. doi: 10.1007/BF02104742. [DOI] [PubMed] [Google Scholar]
  29. Tollervey D. A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 1987 Dec 20;6(13):4169–4175. doi: 10.1002/j.1460-2075.1987.tb02763.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tollervey D., Guthrie C. Deletion of a yeast small nuclear RNA gene impairs growth. EMBO J. 1985 Dec 30;4(13B):3873–3878. doi: 10.1002/j.1460-2075.1985.tb04160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tollervey D., Hurt E. C. The role of small nucleolar ribonucleoproteins in ribosome synthesis. Mol Biol Rep. 1990;14(2-3):103–106. doi: 10.1007/BF00360433. [DOI] [PubMed] [Google Scholar]
  32. Tollervey D., Lehtonen H., Carmo-Fonseca M., Hurt E. C. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 1991 Mar;10(3):573–583. doi: 10.1002/j.1460-2075.1991.tb07984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tollervey D., Mattaj I. W. Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J. 1987 Feb;6(2):469–476. doi: 10.1002/j.1460-2075.1987.tb04777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tyc K., Steitz J. A. U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J. 1989 Oct;8(10):3113–3119. doi: 10.1002/j.1460-2075.1989.tb08463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zagorski J., Tollervey D., Fournier M. J. Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs. Mol Cell Biol. 1988 Aug;8(8):3282–3290. doi: 10.1128/mcb.8.8.3282. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES