Skip to main content
NCBI home page
RNA logoLink to RNA
. 2001 Jun;7(6):896–903. doi: 10.1017/s1355838201010196

Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA.

C A van Beekvelt 1, R E Jeeninga 1, J van't Riet 1, J Venema 1, H A Raué 1
PMCID: PMC1370137  PMID: 11421364

Abstract

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.

Full Text

The Full Text of this article is available as a PDF (321.9 KB).

Selected References

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

  1. Allmang C., Henry Y., Wood H., Morrissey J. P., Petfalski E., Tollervey D. Recognition of cleavage site A(2) in the yeast pre-rRNA. RNA. 1996 Jan;2(1):51–62. [PMC free article] [PubMed] [Google Scholar]
  2. Beltrame M., Henry Y., Tollervey D. Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA. Nucleic Acids Res. 1994 Nov 25;22(23):5139–5147. doi: 10.1093/nar/22.23.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beltrame M., Tollervey D. Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J. 1995 Sep 1;14(17):4350–4356. doi: 10.1002/j.1460-2075.1995.tb00109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beltrame M., Tollervey D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J. 1992 Apr;11(4):1531–1542. doi: 10.1002/j.1460-2075.1992.tb05198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowman L. H., Rabin B., Schlessinger D. Multiple ribosomal RNA cleavage pathways in mammalian cells. Nucleic Acids Res. 1981 Oct 10;9(19):4951–4966. doi: 10.1093/nar/9.19.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavaillé J., Hadjiolov A. A., Bachellerie J. P. Processing of mammalian rRNA precursors at the 3' end of 18S rRNA. Identification of cis-acting signals suggests the involvement of U13 small nucleolar RNA. Eur J Biochem. 1996 Dec 1;242(2):206–213. doi: 10.1111/j.1432-1033.1996.0206r.x. [DOI] [PubMed] [Google Scholar]
  7. Eichler D. C., Craig N. Processing of eukaryotic ribosomal RNA. Prog Nucleic Acid Res Mol Biol. 1994;49:197–239. doi: 10.1016/s0079-6603(08)60051-3. [DOI] [PubMed] [Google Scholar]
  8. Geerlings T. H., Vos J. C., Raué H. A. The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases. RNA. 2000 Dec;6(12):1698–1703. doi: 10.1017/s1355838200001540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  10. Henry Y., Wood H., Morrissey J. P., Petfalski E., Kearsey S., Tollervey D. The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site. EMBO J. 1994 May 15;13(10):2452–2463. doi: 10.1002/j.1460-2075.1994.tb06530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hughes J. M. Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA. J Mol Biol. 1996 Jun 21;259(4):645–654. doi: 10.1006/jmbi.1996.0346. [DOI] [PubMed] [Google Scholar]
  12. Jeeninga R. E., Van Delft Y., de Graaff-Vincent M., Dirks-Mulder A., Venema J., Raué H. A. Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA. RNA. 1997 May;3(5):476–488. [PMC free article] [PubMed] [Google Scholar]
  13. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  14. Kressler D., Linder P., de La Cruz J. Protein trans-acting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Dec;19(12):7897–7912. doi: 10.1128/mcb.19.12.7897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kufel J., Dichtl B., Tollervey D. Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS. RNA. 1999 Jul;5(7):909–917. doi: 10.1017/s135583829999026x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liang W. Q., Fournier M. J. Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2864–2868. doi: 10.1073/pnas.94.7.2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lindahl L., Archer R. H., Zengel J. M. Alternate pathways for processing in the internal transcribed spacer 1 in pre-rRNA of Saccharomyces cerevisiae. Nucleic Acids Res. 1994 Dec 11;22(24):5399–5407. doi: 10.1093/nar/22.24.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Moy T. I., Silver P. A. Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev. 1999 Aug 15;13(16):2118–2133. doi: 10.1101/gad.13.16.2118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mélèse T., Xue Z. The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol. 1995 Jun;7(3):319–324. doi: 10.1016/0955-0674(95)80085-9. [DOI] [PubMed] [Google Scholar]
  20. Peculis B. A., Greer C. L. The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast. RNA. 1998 Dec;4(12):1610–1622. doi: 10.1017/s1355838298981420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raué H. A., Planta R. J. Ribosome biogenesis in yeast. Prog Nucleic Acid Res Mol Biol. 1991;41:89–129. doi: 10.1016/s0079-6603(08)60007-0. [DOI] [PubMed] [Google Scholar]
  22. Raué H. A., Planta R. J. The pathway to maturity: processing of ribosomal RNA in Saccharomyces cerevisiae. Gene Expr. 1995;5(1):71–77. [PMC free article] [PubMed] [Google Scholar]
  23. Sharma K., Tollervey D. Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. Mol Cell Biol. 1999 Sep;19(9):6012–6019. doi: 10.1128/mcb.19.9.6012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stevens A., Hsu C. L., Isham K. R., Larimer F. W. Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1. J Bacteriol. 1991 Nov;173(21):7024–7028. doi: 10.1128/jb.173.21.7024-7028.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Trapman J., Planta R. J. Maturation of ribosomes in yeast. I Kinetic analysis by labelling of high molecular weight rRNA species. Biochim Biophys Acta. 1976 Sep 6;442(3):265–274. doi: 10.1016/0005-2787(76)90301-4. [DOI] [PubMed] [Google Scholar]
  26. Udem S. A., Warner J. R. Ribosomal RNA synthesis in Saccharomyces cerevisiae. J Mol Biol. 1972 Mar 28;65(2):227–242. doi: 10.1016/0022-2836(72)90279-3. [DOI] [PubMed] [Google Scholar]
  27. Venema J., Dirks-Mulder A., Faber A. W., Raué H. A. Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function. Yeast. 1995 Feb;11(2):145–156. doi: 10.1002/yea.320110206. [DOI] [PubMed] [Google Scholar]
  28. Venema J., Henry Y., Tollervey D. Two distinct recognition signals define the site of endonucleolytic cleavage at the 5'-end of yeast 18S rRNA. EMBO J. 1995 Oct 2;14(19):4883–4892. doi: 10.1002/j.1460-2075.1995.tb00169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Venema J., Planta R. J., Raué H. A. In vivo mutational analysis of ribosomal RNA in Saccharomyces cerevisiae. Methods Mol Biol. 1998;77:257–270. doi: 10.1385/0-89603-397-X:257. [DOI] [PubMed] [Google Scholar]
  30. Venema J., Tollervey D. Processing of pre-ribosomal RNA in Saccharomyces cerevisiae. Yeast. 1995 Dec;11(16):1629–1650. doi: 10.1002/yea.320111607. [DOI] [PubMed] [Google Scholar]
  31. Venema J., Tollervey D. RRP5 is required for formation of both 18S and 5.8S rRNA in yeast. EMBO J. 1996 Oct 15;15(20):5701–5714. [PMC free article] [PubMed] [Google Scholar]
  32. Venema J., Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae. Annu Rev Genet. 1999;33:261–311. doi: 10.1146/annurev.genet.33.1.261. [DOI] [PubMed] [Google Scholar]
  33. Warner J. R. Synthesis of ribosomes in Saccharomyces cerevisiae. Microbiol Rev. 1989 Jun;53(2):256–271. doi: 10.1128/mr.53.2.256-271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeh L. C., Thweatt R., Lee J. C. Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common structural motifs. Biochemistry. 1990 Jun 26;29(25):5911–5918. doi: 10.1021/bi00477a005. [DOI] [PubMed] [Google Scholar]
  35. van Beekvelt C. A., Kooi E. A., de Graaff-Vincent M., Riet J., Venema J., Raué H. A. Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation. J Mol Biol. 2000 Feb 11;296(1):7–17. doi: 10.1006/jmbi.1999.3432. [DOI] [PubMed] [Google Scholar]
  36. van Nues R. W., Rientjes J. M., Morré S. A., Mollee E., Planta R. J., Venema J., Raué H. A. Evolutionarily conserved structural elements are critical for processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae precursor ribosomal RNA. J Mol Biol. 1995 Jun 30;250(1):24–36. doi: 10.1006/jmbi.1995.0355. [DOI] [PubMed] [Google Scholar]
  37. van Nues R. W., Rientjes J. M., van der Sande C. A., Zerp S. F., Sluiter C., Venema J., Planta R. J., Raué H. A. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Res. 1994 Mar 25;22(6):912–919. doi: 10.1093/nar/22.6.912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Nues R. W., Venema J., Rientjes J. M., Dirks-Mulder A., Raué H. A. Processing of eukaryotic pre-rRNA: the role of the transcribed spacers. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):789–801. doi: 10.1139/o95-087. [DOI] [PubMed] [Google Scholar]
  39. van der Sande C. A., Kwa M., van Nues R. W., van Heerikhuizen H., Raué H. A., Planta R. J. Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA. J Mol Biol. 1992 Feb 20;223(4):899–910. doi: 10.1016/0022-2836(92)90251-e. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES