Abstract
We constructed a translational fusion between the Saccharomyces cerevisiae actin gene and the Escherichia coli beta-galactosidase structural gene such that expression of beta-galactosidase activity required accurate splicing of the actin intron. Using this chimeric gene, we generated a series of internal deletions which removed the TACTAAC box or, in addition, TACTAAC-like sequences within the intron. Analysis of the fusion transcripts produced in these deletions allowed us to conclude that the TACTAAC-like sequence TACTAAG can substitute, albeit inefficiently, for the authentic TACTAAC box in the splicing process. These results indicate that the yeast splicing machinery can utilize a cryptic TACTAAC box, but there are requirements for primary sequence and proper position.
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Selected References
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- Abelson J. RNA processing and the intervening sequence problem. Annu Rev Biochem. 1979;48:1035–1069. doi: 10.1146/annurev.bi.48.070179.005131. [DOI] [PubMed] [Google Scholar]
- Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
- Gallwitz D. Construction of a yeast actin gene intron deletion mutant that is defective in splicing and leads to the accumulation of precursor RNA in transformed yeast cells. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3493–3497. doi: 10.1073/pnas.79.11.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gallwitz D., Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. doi: 10.1073/pnas.77.5.2546. [DOI] [PMC free article] [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]
- Keller E. B., Noon W. A. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417–7420. doi: 10.1073/pnas.81.23.7417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Käufer N. F., Fried H. M., Schwindinger W. F., Jasin M., Warner J. R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 1983 May 25;11(10):3123–3135. doi: 10.1093/nar/11.10.3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langford C. J., Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. doi: 10.1016/0092-8674(83)90433-6. [DOI] [PubMed] [Google Scholar]
- Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
- Larson G. P., Itakura K., Ito H., Rossi J. J. Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene. Gene. 1983 Apr;22(1):31–39. doi: 10.1016/0378-1119(83)90061-6. [DOI] [PubMed] [Google Scholar]
- Leer R. J., van Raamsdonk-Duin M. M., Hagendoorn M. J., Mager W. H., Planta R. J. Structural comparison of yeast ribosomal protein genes. Nucleic Acids Res. 1984 Sep 11;12(17):6685–6700. doi: 10.1093/nar/12.17.6685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leer R. J., van Raamsdonk-Duin M. M., Molenaar C. M., Cohen L. H., Mager W. H., Planta R. J. The structure of the gene coding for the phosphorylated ribosomal protein S10 in yeast. Nucleic Acids Res. 1982 Oct 11;10(19):5869–5878. doi: 10.1093/nar/10.19.5869. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Mitra G., Warner J. R. A yeast ribosomal protein gene whose intron is in the 5' leader. J Biol Chem. 1984 Jul 25;259(14):9218–9224. [PubMed] [Google Scholar]
- Molenaar C. M., Woudt L. P., Jansen A. E., Mager W. H., Planta R. J., Donovan D. M., Pearson N. J. Structure and organization of two linked ribosomal protein genes in yeast. Nucleic Acids Res. 1984 Oct 11;12(19):7345–7358. doi: 10.1093/nar/12.19.7345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mount S., Steitz J. Lessons from mutant globins. Nature. 1983 Jun 2;303(5916):380–381. doi: 10.1038/303380a0. [DOI] [PubMed] [Google Scholar]
- Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
- Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
- Parker R., Guthrie C. A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation. Cell. 1985 May;41(1):107–118. doi: 10.1016/0092-8674(85)90065-0. [DOI] [PubMed] [Google Scholar]
- Pikielny C. W., Teem J. L., Rosbash M. Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing. Cell. 1983 Sep;34(2):395–403. doi: 10.1016/0092-8674(83)90373-2. [DOI] [PubMed] [Google Scholar]
- Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
- Rodriguez J. R., Pikielny C. W., Rosbash M. In vivo characterization of yeast mRNA processing intermediates. Cell. 1984 Dec;39(3 Pt 2):603–610. doi: 10.1016/0092-8674(84)90467-7. [DOI] [PubMed] [Google Scholar]
- Rosbash M., Harris P. K., Woolford J. L., Jr, Teem J. L. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell. 1981 Jun;24(3):679–686. doi: 10.1016/0092-8674(81)90094-5. [DOI] [PubMed] [Google Scholar]
- Ruskin B., Green M. R. An RNA processing activity that debranches RNA lariats. Science. 1985 Jul 12;229(4709):135–140. doi: 10.1126/science.2990042. [DOI] [PubMed] [Google Scholar]
- Ruskin B., Greene J. M., Green M. R. Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants. Cell. 1985 Jul;41(3):833–844. doi: 10.1016/s0092-8674(85)80064-7. [DOI] [PubMed] [Google Scholar]
- Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
- Teem J. L., Abovich N., Kaufer N. F., Schwindinger W. F., Warner J. R., Levy A., Woolford J., Leer R. J., van Raamsdonk-Duin M. M., Mager W. H. A comparison of yeast ribosomal protein gene DNA sequences. Nucleic Acids Res. 1984 Nov 26;12(22):8295–8312. doi: 10.1093/nar/12.22.8295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Teem J. L., Rosbash M. Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4403–4407. doi: 10.1073/pnas.80.14.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace J. C., Edmonds M. Polyadenylylated nuclear RNA contains branches. Proc Natl Acad Sci U S A. 1983 Feb;80(4):950–954. doi: 10.1073/pnas.80.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zeitlin S., Efstratiadis A. In vivo splicing products of the rabbit beta-globin pre-mRNA. Cell. 1984 Dec;39(3 Pt 2):589–602. doi: 10.1016/0092-8674(84)90466-5. [DOI] [PubMed] [Google Scholar]