Abstract
We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.
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Selected References
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- Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
- Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
- Bevan E. A., Herring A. J., Mitchell D. J. Preliminary characterization of two species of dsRNA in yeast and their relationship to the "killer" character. Nature. 1973 Sep 14;245(5420):81–86. doi: 10.1038/245081b0. [DOI] [PubMed] [Google Scholar]
- Burgers P. M., Percival K. J. Transformation of yeast spheroplasts without cell fusion. Anal Biochem. 1987 Jun;163(2):391–397. doi: 10.1016/0003-2697(87)90240-5. [DOI] [PubMed] [Google Scholar]
- Delorme E. Transformation of Saccharomyces cerevisiae by electroporation. Appl Environ Microbiol. 1989 Sep;55(9):2242–2246. doi: 10.1128/aem.55.9.2242-2246.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fujimura T., Wickner R. B. Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain. Cell. 1988 Nov 18;55(4):663–671. doi: 10.1016/0092-8674(88)90225-5. [DOI] [PubMed] [Google Scholar]
- Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Kleinschmidt A. M., Pederson T. RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1283–1287. doi: 10.1073/pnas.87.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klump W. M., Bergmann I., Müller B. C., Ameis D., Kandolf R. Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA: two initial 5' uridine residues are regained during plus-strand RNA synthesis. J Virol. 1990 Apr;64(4):1573–1583. doi: 10.1128/jvi.64.4.1573-1583.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malone R. W., Felgner P. L., Verma I. M. Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6077–6081. doi: 10.1073/pnas.86.16.6077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Nguyen V. T., Morange M., Bensaude O. Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells. Anal Biochem. 1988 Jun;171(2):404–408. doi: 10.1016/0003-2697(88)90505-2. [DOI] [PubMed] [Google Scholar]
- Ow D. W., DE Wet J. R., Helinski D. R., Howell S. H., Wood K. V., Deluca M. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science. 1986 Nov 14;234(4778):856–859. doi: 10.1126/science.234.4778.856. [DOI] [PubMed] [Google Scholar]
- Sarnow P. Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. J Virol. 1989 Jan;63(1):467–470. doi: 10.1128/jvi.63.1.467-470.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sarnow P. Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. J Virol. 1989 Jan;63(1):467–470. doi: 10.1128/jvi.63.1.467-470.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simoes E. A., Sarnow P. An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells. J Virol. 1991 Feb;65(2):913–921. doi: 10.1128/jvi.65.2.913-921.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sommer S. S., Wickner R. B. Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes [HOK], [NEX] and [EXL] are on one of these dsRNAs. Cell. 1982 Dec;31(2 Pt 1):429–441. doi: 10.1016/0092-8674(82)90136-2. [DOI] [PubMed] [Google Scholar]
- Sompayrac L. M., Danna K. J. Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7575–7578. doi: 10.1073/pnas.78.12.7575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sonenberg N., Pelletier J. Poliovirus translation: a paradigm for a novel initiation mechanism. Bioessays. 1989 Nov;11(5):128–132. doi: 10.1002/bies.950110504. [DOI] [PubMed] [Google Scholar]
- Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tipper D. J., Bostian K. A. Double-stranded ribonucleic acid killer systems in yeasts. Microbiol Rev. 1984 Jun;48(2):125–156. doi: 10.1128/mr.48.2.125-156.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaheri A., Pagano J. S. Infectious poliovirus RNA: a sensitive method of assay. Virology. 1965 Nov;27(3):434–436. doi: 10.1016/0042-6822(65)90126-1. [DOI] [PubMed] [Google Scholar]
- Vodkin M., Katterman F., Fink G. R. Yeast killer mutants with altered double-stranded ribonucleic acid. J Bacteriol. 1974 Feb;117(2):681–686. doi: 10.1128/jb.117.2.681-686.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss B., Nitschko H., Ghattas I., Wright R., Schlesinger S. Evidence for specificity in the encapsidation of Sindbis virus RNAs. J Virol. 1989 Dec;63(12):5310–5318. doi: 10.1128/jvi.63.12.5310-5318.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wesolowski M., Wickner R. B. Two new double-stranded RNA molecules showing non-mendelian inheritance and heat inducibility in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):181–187. doi: 10.1128/mcb.4.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wickner R. B. Double-stranded RNA replication in yeast: the killer system. Annu Rev Biochem. 1986;55:373–395. doi: 10.1146/annurev.bi.55.070186.002105. [DOI] [PubMed] [Google Scholar]
- Wickner R. B. Yeast virology. FASEB J. 1989 Sep;3(11):2257–2265. doi: 10.1096/fasebj.3.11.2550303. [DOI] [PubMed] [Google Scholar]
- Zlotnik H., Fernandez M. P., Bowers B., Cabib E. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol. 1984 Sep;159(3):1018–1026. doi: 10.1128/jb.159.3.1018-1026.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2330–2334. doi: 10.1073/pnas.83.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]