Abstract
In yeast, the 5' end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5' region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.
Full Text
The Full Text of this article is available as a PDF (506.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alksne L. E., Anthony R. A., Liebman S. W., Warner J. R. An accuracy center in the ribosome conserved over 2 billion years. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9538–9541. doi: 10.1073/pnas.90.20.9538. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alksne L. E., Warner J. R. A novel cloning strategy reveals the gene for the yeast homologue to Escherichia coli ribosomal protein S12. J Biol Chem. 1993 May 25;268(15):10813–10819. [PubMed] [Google Scholar]
- All-Robyn J. A., Brown N., Otaka E., Liebman S. W. Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein. Mol Cell Biol. 1990 Dec;10(12):6544–6553. doi: 10.1128/mcb.10.12.6544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Dammel C. S., Noller H. F. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 1993 Apr;7(4):660–670. doi: 10.1101/gad.7.4.660. [DOI] [PubMed] [Google Scholar]
- Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Nogi Y., Yano R., Dodd J., Carles C., Nomura M. Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures. Mol Cell Biol. 1993 Jan;13(1):114–122. doi: 10.1128/mcb.13.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nogi Y., Yano R., Nomura M. Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3962–3966. doi: 10.1073/pnas.88.9.3962. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petfalski E., Dandekar T., Henry Y., Tollervey D. Processing of the precursors to small nucleolar RNAs and rRNAs requires common components. Mol Cell Biol. 1998 Mar;18(3):1181–1189. doi: 10.1128/mcb.18.3.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Poot R. A., Pleij C. W., van Duin J. The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation. Nucleic Acids Res. 1996 Oct 1;24(19):3670–3676. doi: 10.1093/nar/24.19.3670. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharma K., Fabre E., Tekotte H., Hurt E. C., Tollervey D. Yeast nucleoporin mutants are defective in pre-tRNA splicing. Mol Cell Biol. 1996 Jan;16(1):294–301. doi: 10.1128/mcb.16.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Vincent A., Liebman S. W. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein. Genetics. 1992 Oct;132(2):375–386. doi: 10.1093/genetics/132.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]