Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Apr;17(4):1959–1965. doi: 10.1128/mcb.17.4.1959

Ribosomal protein L32 of Saccharomyces cerevisiae influences both the splicing of its own transcript and the processing of rRNA.

J Vilardell 1, J R Warner 1
PMCID: PMC232042  PMID: 9121443

Abstract

Ribosomal protein L32 of Saccharomyces cerevisiae binds to and regulates the splicing and the translation of the transcript of its own gene. Selecting for mutants deficient in the regulation of splicing, we have identified a mutant form of L32 that no longer binds to the transcript of RPL32 and therefore does not regulate its splicing. The mutation is the deletion of an isoleucine residue from a highly conserved hydrophobic domain near the middle of L32. The mutant protein supports growth, at a reduced rate, and is found at normal levels in mature ribosomes. However, in cells homozygous for the mutant gene, the rate of processing of the ribosomal RNA component of the 60S ribosomal subunit is severely reduced, leading to an insufficiency of 60S subunits. L32 must be considered a remarkable protein. Composed of only 104 amino acids, it appears to interact with three distinct RNA molecules to influence three different elements of RNA processing and function in three different locations of the cell: the processing of pre-rRNA in the nucleolus, the splicing of the RPL32 transcript in the nucleus, and the translation of the spliced RPL32 mRNA in the cytoplasm.

Full Text

The Full Text of this article is available as a PDF (957.7 KB).

Selected References

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

  1. Battiste J. L., Mao H., Rao N. S., Tan R., Muhandiram D. R., Kay L. E., Frankel A. D., Williamson J. R. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science. 1996 Sep 13;273(5281):1547–1551. doi: 10.1126/science.273.5281.1547. [DOI] [PubMed] [Google Scholar]
  2. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  3. Caponigro G., Parker R. Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae. Microbiol Rev. 1996 Mar;60(1):233–249. doi: 10.1128/mr.60.1.233-249.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dabeva M. D., Post-Beittenmiller M. A., Warner J. R. Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5854–5857. doi: 10.1073/pnas.83.16.5854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dabeva M. D., Warner J. R. Ribosomal protein L32 of Saccharomyces cerevisiae regulates both splicing and translation of its own transcript. J Biol Chem. 1993 Sep 15;268(26):19669–19674. [PubMed] [Google Scholar]
  6. Dabeva M. D., Warner J. R. The yeast ribosomal protein L32 and its gene. J Biol Chem. 1987 Nov 25;262(33):16055–16059. [PubMed] [Google Scholar]
  7. Draper D. E., Deckman I. C., Vartikar J. V. Physical studies of ribosomal protein-RNA interactions. Methods Enzymol. 1988;164:203–220. doi: 10.1016/s0076-6879(88)64044-4. [DOI] [PubMed] [Google Scholar]
  8. Draper D. E. Protein-RNA recognition. Annu Rev Biochem. 1995;64:593–620. doi: 10.1146/annurev.bi.64.070195.003113. [DOI] [PubMed] [Google Scholar]
  9. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  10. Eng F. J., Warner J. R. Structural basis for the regulation of splicing of a yeast messenger RNA. Cell. 1991 May 31;65(5):797–804. doi: 10.1016/0092-8674(91)90387-e. [DOI] [PubMed] [Google Scholar]
  11. Ge H., Manley J. L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. doi: 10.1016/0092-8674(90)90236-8. [DOI] [PubMed] [Google Scholar]
  12. Gutell R. R., Gray M. W., Schnare M. N. A compilation of large subunit (23S and 23S-like) ribosomal RNA structures: 1993. Nucleic Acids Res. 1993 Jul 1;21(13):3055–3074. doi: 10.1093/nar/21.13.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoffman D. W., Query C. C., Golden B. L., White S. W., Keene J. D. RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2495–2499. doi: 10.1073/pnas.88.6.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Imai Y., Matsushima Y., Sugimura T., Terada M. A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res. 1991 May 25;19(10):2785–2785. doi: 10.1093/nar/19.10.2785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kanaar R., Lee A. L., Rudner D. Z., Wemmer D. E., Rio D. C. Interaction of the sex-lethal RNA binding domains with RNA. EMBO J. 1995 Sep 15;14(18):4530–4539. doi: 10.1002/j.1460-2075.1995.tb00132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolodrubetz D., Burgum A. Sequence and genetic analysis of NHP2: a moderately abundant high mobility group-like nuclear protein with an essential function in Saccharomyces cerevisiae. Yeast. 1991 Feb;7(2):79–90. doi: 10.1002/yea.320070202. [DOI] [PubMed] [Google Scholar]
  17. Krainer A. R., Conway G. C., Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. doi: 10.1016/0092-8674(90)90237-9. [DOI] [PubMed] [Google Scholar]
  18. Kumar A., Warner J. R. Characterization of ribosomal precursor particles from HeLa cell nucleoli. J Mol Biol. 1972 Jan 28;63(2):233–246. doi: 10.1016/0022-2836(72)90372-5. [DOI] [PubMed] [Google Scholar]
  19. Lesser C. F., Guthrie C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science. 1993 Dec 24;262(5142):1982–1988. doi: 10.1126/science.8266093. [DOI] [PubMed] [Google Scholar]
  20. Levy D., Larner A., Chaudhuri A., Babiss L. E., Darnell J. E., Jr Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8929–8933. doi: 10.1073/pnas.83.23.8929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li B., Vilardell J., Warner J. R. An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1596–1600. doi: 10.1073/pnas.93.4.1596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li H., Dalal S., Kohler J., Vilardell J., White S. A. Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop. J Mol Biol. 1995 Jul 21;250(4):447–459. doi: 10.1006/jmbi.1995.0389. [DOI] [PubMed] [Google Scholar]
  23. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moritz M., Paulovich A. G., Tsay Y. F., Woolford J. L., Jr Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2261–2274. doi: 10.1083/jcb.111.6.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moritz M., Pulaski B. A., Woolford J. L., Jr Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16. Mol Cell Biol. 1991 Nov;11(11):5681–5692. doi: 10.1128/mcb.11.11.5681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nagai K., Oubridge C., Ito N., Avis J., Evans P. The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA. Trends Biochem Sci. 1995 Jun;20(6):235–240. doi: 10.1016/s0968-0004(00)89024-6. [DOI] [PubMed] [Google Scholar]
  27. Powers T., Daubresse G., Noller H. F. Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits. J Mol Biol. 1993 Jul 20;232(2):362–374. doi: 10.1006/jmbi.1993.1396. [DOI] [PubMed] [Google Scholar]
  28. Rost B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996;266:525–539. doi: 10.1016/s0076-6879(96)66033-9. [DOI] [PubMed] [Google Scholar]
  29. Rotenberg M. O., Moritz M., Woolford J. L., Jr Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes. Genes Dev. 1988 Feb;2(2):160–172. doi: 10.1101/gad.2.2.160. [DOI] [PubMed] [Google Scholar]
  30. Roth H. E., Nierhaus K. H. Assembly map of the 50-S subunit from Escherichia coli ribosomes, covering the proteins present in the first reconstitution intermediate particle. Eur J Biochem. 1980 Jan;103(1):95–98. doi: 10.1111/j.1432-1033.1980.tb04292.x. [DOI] [PubMed] [Google Scholar]
  31. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  32. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  35. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  37. Trapman J., Retèl J., Planta R. J. Ribosomal precursor particles from yeast. Exp Cell Res. 1975 Jan;90(1):95–104. doi: 10.1016/0014-4827(75)90361-4. [DOI] [PubMed] [Google Scholar]
  38. Traub P., Nomura M. Studies on the assembly of ribosomes in vitro. Cold Spring Harb Symp Quant Biol. 1969;34:63–67. doi: 10.1101/sqb.1969.034.01.010. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Valcárcel J., Green M. R. The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci. 1996 Aug;21(8):296–301. [PubMed] [Google Scholar]
  41. Valcárcel J., Green M. R. The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci. 1996 Aug;21(8):296–301. [PubMed] [Google Scholar]
  42. 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]
  43. Vilardell J., Warner J. R. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes Dev. 1994 Jan;8(2):211–220. doi: 10.1101/gad.8.2.211. [DOI] [PubMed] [Google Scholar]
  44. Warner J. R., Mitra G., Schwindinger W. F., Studeny M., Fried H. M. Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover. Mol Cell Biol. 1985 Jun;5(6):1512–1521. doi: 10.1128/mcb.5.6.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Warner J. R., Morgan S. A., Shulman R. W. Kinetics of labeling of the S-adenosylmethionine pool of Saccharomyces cerevisiae. J Bacteriol. 1976 Mar;125(3):887–891. doi: 10.1128/jb.125.3.887-891.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Warner J. R., Soeiro R. Nascent ribosomes from HeLa cells. Proc Natl Acad Sci U S A. 1967 Nov;58(5):1984–1990. doi: 10.1073/pnas.58.5.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. White S. A., Li H. Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition. RNA. 1996 Mar;2(3):226–234. [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES