Abstract
Using a series of point mutations in chimeric reporter gene constructs consisting of the 5' regions of the Chlamydomonas chloroplast rbcL or atpB genes fused 5' to the coding sequence of the bacterial uidA (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 5' untranslated regions (5' UTRs) of transcripts of the chloroplast genes rbcL and atpB in Chlamydomonas reinhardtii. In chimeric rbcL 5' UTR:GUS transcripts, replacement of single nucleotides in the 10-nt sequence 5'-AUUUCCGGAC-3', extending from positions +38 to +47 relative to the transcripts' 5' terminus, shortened transcript longevity and led to a reduction in transcript abundance of more than 95%. A similar mutational analysis of atpB 5' UTR:GUS transcripts showed that the 12-nt atpB 5' UTR sequence 5'-AUAAGCGUUAGU-3', extending from position +31 to position +42, is important for transcript stability and transcript accumulation in the chloroplast of Chlamydomonas. We discuss how the 5' UTR sequence elements, which are predicted to be part of RNA secondary structures, might function in RNA stabilization.
Full Text
The Full Text of this article is available as a PDF (466.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arnold T. E., Yu J., Belasco J. G. mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation. RNA. 1998 Mar;4(3):319–330. [PMC free article] [PubMed] [Google Scholar]
- Blowers A. D., Bogorad L., Shark K. B., Sanford J. C. Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome. Plant Cell. 1989 Jan;1(1):123–132. doi: 10.1105/tpc.1.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blowers A. D., Ellmore G. S., Klein U., Bogorad L. Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell. 1990 Nov;2(11):1059–1070. doi: 10.1105/tpc.2.11.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blowers A. D., Klein U., Ellmore G. S., Bogorad L. Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes. Mol Gen Genet. 1993 Apr;238(3):339–349. doi: 10.1007/BF00291992. [DOI] [PubMed] [Google Scholar]
- Boudreau E., Nickelsen J., Lemaire S. D., Ossenbühl F., Rochaix J. D. The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability. EMBO J. 2000 Jul 3;19(13):3366–3376. doi: 10.1093/emboj/19.13.3366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bouvet P., Belasco J. G. Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli. Nature. 1992 Dec 3;360(6403):488–491. doi: 10.1038/360488a0. [DOI] [PubMed] [Google Scholar]
- Chen L. H., Emory S. A., Bricker A. L., Bouvet P., Belasco J. G. Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA. J Bacteriol. 1991 Aug;173(15):4578–4586. doi: 10.1128/jb.173.15.4578-4586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Douglas S. E., Penny S. L. The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae. J Mol Evol. 1999 Feb;48(2):236–244. doi: 10.1007/pl00006462. [DOI] [PubMed] [Google Scholar]
- Drager R. G., Girard-Bascou J., Choquet Y., Kindle K. L., Stern D. B. In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA. Plant J. 1998 Jan;13(1):85–96. doi: 10.1046/j.1365-313x.1998.00016.x. [DOI] [PubMed] [Google Scholar]
- Drager R. G., Higgs D. C., Kindle K. L., Stern D. B. 5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways. Plant J. 1999 Sep;19(5):521–531. doi: 10.1046/j.1365-313x.1999.00546.x. [DOI] [PubMed] [Google Scholar]
- Drapier D., Girard-Bascou J., Wollman F. A. Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas. Plant Cell. 1992 Mar;4(3):283–295. doi: 10.1105/tpc.4.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dron M., Rahire M., Rochaix J. D. Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the gene of the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes. J Mol Biol. 1982 Dec 25;162(4):775–793. doi: 10.1016/0022-2836(82)90547-2. [DOI] [PubMed] [Google Scholar]
- Eibl C, Zou Z, Beck a, Kim M, Mullet J, Koop HU. In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J. 1999 Aug;19(3):333–345. doi: 10.1046/j.1365-313x.1999.00543.x. [DOI] [PubMed] [Google Scholar]
- Emory S. A., Bouvet P., Belasco J. G. A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli. Genes Dev. 1992 Jan;6(1):135–148. doi: 10.1101/gad.6.1.135. [DOI] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Grunberg-Manago M. Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annu Rev Genet. 1999;33:193–227. doi: 10.1146/annurev.genet.33.1.193. [DOI] [PubMed] [Google Scholar]
- Hauser C. R., Gillham N. W., Boynton J. E. Translational regulation of chloroplast genes. Proteins binding to the 5'-untranslated regions of chloroplast mRNAs in Chlamydomonas reinhardtii. J Biol Chem. 1996 Jan 19;271(3):1486–1497. doi: 10.1074/jbc.271.3.1486. [DOI] [PubMed] [Google Scholar]
- Hayes R., Kudla J., Gruissem W. Degrading chloroplast mRNA: the role of polyadenylation. Trends Biochem Sci. 1999 May;24(5):199–202. doi: 10.1016/s0968-0004(99)01388-2. [DOI] [PubMed] [Google Scholar]
- Higgs D. C., Shapiro R. S., Kindle K. L., Stern D. B. Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation. Mol Cell Biol. 1999 Dec;19(12):8479–8491. doi: 10.1128/mcb.19.12.8479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hotchkiss T. L., Hollingsworth M. J. ATP synthase 5' untranslated regions are specifically bound by chloroplast polypeptides. Curr Genet. 1999 Jun;35(5):512–520. doi: 10.1007/s002940050447. [DOI] [PubMed] [Google Scholar]
- Hwang S., Kawazoe R., Herrin D. L. Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):996–1000. doi: 10.1073/pnas.93.3.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnston S. A., Anziano P. Q., Shark K., Sanford J. C., Butow R. A. Mitochondrial transformation in yeast by bombardment with microprojectiles. Science. 1988 Jun 10;240(4858):1538–1541. doi: 10.1126/science.2836954. [DOI] [PubMed] [Google Scholar]
- Klein R. R., Mullet J. E. Control of gene expression during higher plant chloroplast biogenesis. Protein synthesis and transcript levels of psbA, psaA-psaB, and rbcL in dark-grown and illuminated barley seedlings. J Biol Chem. 1987 Mar 25;262(9):4341–4348. [PubMed] [Google Scholar]
- Klein U., De Camp J. D., Bogorad L. Two types of chloroplast gene promoters in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3453–3457. doi: 10.1073/pnas.89.8.3453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klein U., Salvador M. L., Bogorad L. Activity of the Chlamydomonas chloroplast rbcL gene promoter is enhanced by a remote sequence element. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10819–10823. doi: 10.1073/pnas.91.23.10819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Komine Y., Kwong L., Anguera M. C., Schuster G., Stern D. B. Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii. RNA. 2000 Apr;6(4):598–607. doi: 10.1017/s1355838200992252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuchka M. R., Goldschmidt-Clermont M., van Dillewijn J., Rochaix J. D. Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II. Cell. 1989 Sep 8;58(5):869–876. doi: 10.1016/0092-8674(89)90939-2. [DOI] [PubMed] [Google Scholar]
- Lisitsky I., Klaff P., Schuster G. Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13398–13403. doi: 10.1073/pnas.93.23.13398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mackie G. A. Stabilization of circular rpsT mRNA demonstrates the 5'-end dependence of RNase E action in vivo. J Biol Chem. 2000 Aug 18;275(33):25069–25072. doi: 10.1074/jbc.C000363200. [DOI] [PubMed] [Google Scholar]
- Merchant S., Bogorad L. Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi. Mol Cell Biol. 1986 Feb;6(2):462–469. doi: 10.1128/mcb.6.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
- Nickelsen J., Fleischmann M., Boudreau E., Rahire M., Rochaix J. D. Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas. Plant Cell. 1999 May;11(5):957–970. doi: 10.1105/tpc.11.5.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nickelsen J. Mutations at three different nuclear loci of Chlamydomonas suppress a defect in chloroplast psbD mRNA accumulation. Curr Genet. 2000 Feb;37(2):136–142. doi: 10.1007/s002940050020. [DOI] [PubMed] [Google Scholar]
- Nickelsen J. Transcripts containing the 5' untranslated regions of the plastid genes psbA and psbB from higher plants are unstable in Chlamydomonas reinhardtii chloroplasts. Mol Gen Genet. 1999 Dec;262(4-5):768–771. doi: 10.1007/s004380051139. [DOI] [PubMed] [Google Scholar]
- Nickelsen J., van Dillewijn J., Rahire M., Rochaix J. D. Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. EMBO J. 1994 Jul 1;13(13):3182–3191. doi: 10.1002/j.1460-2075.1994.tb06617.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rauhut R., Klug G. mRNA degradation in bacteria. FEMS Microbiol Rev. 1999 Jun;23(3):353–370. doi: 10.1111/j.1574-6976.1999.tb00404.x. [DOI] [PubMed] [Google Scholar]
- Salvador M. L., Klein U., Bogorad L. 5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1556–1560. doi: 10.1073/pnas.90.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Salvador M. L., Klein U., Bogorad L. Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation. Plant J. 1993 Feb;3(2):213–219. doi: 10.1046/j.1365-313x.1993.t01-13-00999.x. [DOI] [PubMed] [Google Scholar]
- Salvador M. L., Klein U. The redox state regulates RNA degradation in the chloroplast of Chlamydomonas reinhardtii. Plant Physiol. 1999 Dec;121(4):1367–1374. doi: 10.1104/pp.121.4.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sarkar N. Polyadenylation of mRNA in prokaryotes. Annu Rev Biochem. 1997;66:173–197. doi: 10.1146/annurev.biochem.66.1.173. [DOI] [PubMed] [Google Scholar]
- Shiina T., Allison L., Maliga P. rbcL Transcript levels in tobacco plastids are independent of light: reduced dark transcription rate is compensated by increased mRNA stability. Plant Cell. 1998 Oct;10(10):1713–1722. doi: 10.1105/tpc.10.10.1713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sieburth L. E., Berry-Lowe S., Schmidt G. W. Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants. Plant Cell. 1991 Feb;3(2):175–189. doi: 10.1105/tpc.3.2.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singh M., Boutanaev A., Zucchi P., Bogorad L. Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts. Proc Natl Acad Sci U S A. 2001 Feb 13;98(5):2289–2294. doi: 10.1073/pnas.041609798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sueoka N. MITOTIC REPLICATION OF DEOXYRIBONUCLEIC ACID IN CHLAMYDOMONAS REINHARDI. Proc Natl Acad Sci U S A. 1960 Jan;46(1):83–91. doi: 10.1073/pnas.46.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turmel M., Otis C., Lemieux C. The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes. Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10248–10253. doi: 10.1073/pnas.96.18.10248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaistij F. E., Goldschmidt-Clermont M., Wostrikoff K., Rochaix J. D. Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas reinhardtii. Plant J. 2000 Mar;21(5):469–482. doi: 10.1046/j.1365-313x.2000.00700.x. [DOI] [PubMed] [Google Scholar]
- Woessner J. P., Gillham N. W., Boynton J. E. The sequence of the chloroplast atpB gene and its flanking regions in Chlamydomonas reinhardtii. Gene. 1986;44(1):17–28. doi: 10.1016/0378-1119(86)90038-7. [DOI] [PubMed] [Google Scholar]