Abstract
Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5′- or 3′-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5′- and 3′-UTRs. In general, the 5′-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5′-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3′-UTR had little impact on mRNA and protein accumulation, as long as a 3′-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5′-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allison LA (2000) The role of sigma factors in plastid transcription. Biochimie 82:537–548
Anthonisen IL, Salvador ML, Klein U (2001) Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii. RNA 7:1024–1033
Auchincloss AH, Zerges W, Perron K, Girard-Bascou J, Rochaix JD (2002) Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii. J Cell Biol 157:953–962
Barkan A, Goldschmidt-Clermont M (2000) Participation of nuclear genes in chloroplast gene expression. Biochimie 82:559–572
Blowers AD, Ellmore GS, Klein U, Bogorad L (1990) Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell 2:1059–1070
Blowers AD, Klein U, Ellmore GS, Bogorad L (1993) Functional in vivo analyses of the 3′ flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes. Mol Gen Genet 238:339–349
Boudreau E, Nickelsen J, Lemaire SD, Ossenbuhl F, Rochaix JD (2000) The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability. Embo J 19:3366–3376
Bruick RK, Mayfield SP (1999) Light-activated translation of chloroplast mRNAs. Trends Plant Sci 4:190–195
Choquet Y, Wollman FA (2002) Translational regulations as specific traits of chloroplast gene expression. FEBS Lett 529:39–42
Cohen A, Yohn CB, Bruick RK, Mayfield SP (1998) Translational regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Methods Enzymol 297:192–208
Dauvillee D, Stampacchia O, Girard-Bascou J, Rochaix JD (2003) Tab2 is a novel conserved RNA binding protein required for translation of the chloroplast psaB mRNA. Embo J 22:6378–6388
Eberhard S, Drapier D, Wollman FA (2002) Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. Plant J 31:149–160
Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU (1999) 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 19:333–345
Fargo DC, Zhang M, Gillham NW, Boynton JE (1998) Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli. Mol Gen Genet 257:271–282
Fisk DG, Walker MB, Barkan A (1999) Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression. Embo J 18:2621–2630
Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30:733–744
Gallie DR (1998) A tale of two termini: a functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation. Gene 216:1–11
Goldschmidt-Clermont M (1998) Coordination of nuclear and chloroplast gene expression in plant cells. Int Rev Cytol 177:115–180
Hayes R, Kudla J, Schuster G, Gabay L, Maliga P, Gruissem W (1996) Chloroplast mRNA 3′-end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins. Embo J 15:1132–1141
Herrin DL, Michaels AS, Paul AL (1986) Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol 103:1837–1845
Higgs DC, Shapiro RS, Kindle KL, Stern DB (1999) Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation. Mol Cell Biol 19:8479–8491
Hirose T, Sugiura M (2004) Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs. Plant Cell Physiol 45:114–117
Ishikura K, Takaoka Y, Kato K, Sekine M, Yoshida K, Shinmyo A (1999) Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast. J Biosci Bioeng 87:307–314
Kasai S, Yoshimura S, Ishikura K, Takaoka Y, Kobayashi K, Kato K, Shinmyo A (2003) Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii. J Biosci Bioeng 95:276–282
Katz YS, Danon A (2002) The 3′-untranslated region of chloroplast psbA mRNA stabilizes binding of regulatory proteins to the leader of the message. J Biol Chem 277:18665–18669
Kim J, Mullet JE (1994) Ribosome-binding sites on chloroplast rbcL and psbA mRNAs and light-induced initiation of D1 translation. Plant Mol Biol 25:437–448
Klein U, Salvador ML, Bogorad L (1994) Activity of the Chlamydomonas chloroplast rbcL gene promoter is enhanced by a remote sequence element. Proc Natl Acad Sci U S A 91:10819–10823
Kuroda H, Maliga P (2001) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125:430–436
Malnoe P, Mayfield SP, Rochaix JD (1988) Comparative analysis of the biogenesis of photosystem II in the wild-type and Y-1 mutant of Chlamydomonas reinhardtii. J Cell Biol 106:609–616
Mayfield SP, Cohen A, Danon A, Yohn CB (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5′ untranslated region. J Cell Biol 127:1537–1545
Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 100:438–442
Mayfield SP, Schultz J (2004) Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J 37:449–458
Meierhoff K, Felder S, Nakamura T, Bechtold N, Schuster G (2003) HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs. Plant Cell 15:1480–1495
Memon AR, Meng B, Mullet JE (1996) RNA-binding proteins of 37/38 kDA bind specifically to the barley chloroplast psbA 3′-end untranslated RNA. Plant Mol Biol 30:1195–1205
Monde RA, Schuster G, Stern DB (2000) Processing and degradation of chloroplast mRNA. Biochimie 82:573–582
Nickelsen J, Fleischmann M, Boudreau E, Rahire M, Rochaix JD (1999) Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas. Plant Cell 11:957–970
Ossenbuhl F, Nickelsen J (2000) cis- and trans-Acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii. Mol Cell Biol 20:8134–8142
Rattanachaikunsopon P, Rosch C, Kuchka MR (1999) Cloning and characterization of the nuclear AC115 gene of Chlamydomonas reinhardtii. Plant Mol Biol 39:1–10
Reinbothe S, Reinbothe C, Heintzen C, Seidenbecher C, Parthier B (1993) A methyl jasmonate-induced shift in the length of the 5′ untranslated region impairs translation of the plastid rbcL transcript in barley. Embo J 12:1505–1512
Rochaix JD (1996) Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Plant Mol Biol 32:327–341
Rott R, Liveanu V, Drager RG, Stern DB, Schuster G (1998) The sequence and structure of the 3′-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability. Plant Mol Biol 36:307–314
Salvador ML, Klein U, Bogorad L (1993) 5′ sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts. Proc Natl Acad Sci U S A 90:1556–1560
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Singh M, Boutanaev A, Zucchi P, Bogorad L (2001) Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts. Proc Natl Acad Sci U S A 98:2289–2294
Somanchi A, Barnes D, Mayfield SP (2005) A nuclear gene of Chlamydomonas reinhardtii, tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA. Plant J 42:341–352
Staub JM, Maliga P (1993) Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. Embo J 12:601–606
Stern DB, Radwanski ER, Kindle KL (1991) A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3:285–297
Sugita M, Sugiura M (1996) Regulation of gene expression in chloroplasts of higher plants. Plant Mol Biol 32:315–326
Sugiura M, Hirose T, Sugita M (1998) Evolution and mechanism of translation in chloroplasts. Annu Rev Genet 32:437–459
Vaistij FE, Boudreau E, Lemaire SD, Goldschmidt-Clermont M, Rochaix JD (2000) Characterization of Mbb1, a nucleus-encoded tetratricopeptide-like repeat protein required for expression of the chloroplast psbB/psbT/psbH gene cluster in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 97:14813–14818
Wells SE, Hillner PE, Vale RD, Sachs AB (1998) Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 2:135–140
Zerges W (2000) Translation in chloroplasts. Biochimie 82:583–601
Zou Z, Eibl C, Koop HU (2003) The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency. Mol Genet Genomics 269:340–349
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Acknowledgements
We thank Veronica Beligni for a critique of the manuscript. This work was supported by funds from the U.S. Department of Energy (93ER70116) and the National Institutes of Health (GM54659). Dwight Barnes was supported by Bauce Foundation and Skaggs Fellowships. The work has been carried out in compliance with the current laws governing genetic experimentation in the USA.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by R. Herrmann
Rights and permissions
About this article
Cite this article
Barnes, D., Franklin, S., Schultz, J. et al. Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Genet Genomics 274, 625–636 (2005). https://doi.org/10.1007/s00438-005-0055-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-005-0055-y