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The 3′-Untranslated Region of Chloroplast psbA mRNA Stabilizes Binding of Regulatory Proteins to the Leader of the Message

2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês

10.1074/jbc.m201033200

ISSN

1083-351X

Autores

Yael S. Katz, Avihai Danon,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

The 5′-leader and 3′-tail of chloroplast mRNAs have been suggested to play a role in posttranscriptional regulation of expression of the message. The regulation is thought to be mediated, at least in part, by regulatory proteins that are encoded by the nuclear genome and targeted to the chloroplast where they interact with chloroplast mRNAs. Previous studies identified high affinity binding of the 5′-untranslated region (UTR) of the chloroplast psbA mRNA by Chlamydomonas reinhardtii proteins. Here we tested whether the 3′-UTR of psbA mRNA alone or linked in cis with the 5′-UTR of the mRNA affects the high affinity binding of the message in vitro. We did not detect high affinity binding that is unique to the 3′-UTR. However, we show that the cis-linked 3′-UTR increases the stability of the 5′-UTR binding complex. This effect could provide a means for translational discrimination against mRNAs that are incorrectly processed. The 5′-leader and 3′-tail of chloroplast mRNAs have been suggested to play a role in posttranscriptional regulation of expression of the message. The regulation is thought to be mediated, at least in part, by regulatory proteins that are encoded by the nuclear genome and targeted to the chloroplast where they interact with chloroplast mRNAs. Previous studies identified high affinity binding of the 5′-untranslated region (UTR) of the chloroplast psbA mRNA by Chlamydomonas reinhardtii proteins. Here we tested whether the 3′-UTR of psbA mRNA alone or linked in cis with the 5′-UTR of the mRNA affects the high affinity binding of the message in vitro. We did not detect high affinity binding that is unique to the 3′-UTR. However, we show that the cis-linked 3′-UTR increases the stability of the 5′-UTR binding complex. This effect could provide a means for translational discrimination against mRNAs that are incorrectly processed. In initiation of translation, ribosomes bind to the 5′-untranslated region (UTR) 1The abbreviations used are: UTRuntranslated regionGMSgel mobility shiftUVCLUV-cross-linkingrrecombinant 1The abbreviations used are: UTRuntranslated regionGMSgel mobility shiftUVCLUV-cross-linkingrrecombinant of the mRNA and are consequently directed to the initiator codon of the open reading frame. The 5′-UTR of several chloroplast mRNAs in higher plants and in the unicellular green alga Chlamydomonas reinhardtii contain regions of inverted repeats and binding sites for nuclear encoded proteins. These interactions have been shown to be involved not only in regulation of translation but also in processing and stability of the message (1Alexander C. Faber N. Klaff P. Nucleic Acids Res. 1998; 26: 2265-2272Crossref PubMed Scopus (44) Google Scholar, 2Boudreau E. Nickelsen J. Lemaire S.D. Ossenbuhl F. Rochaix J.D. EMBO J. 2000; 19: 3366-3376Crossref PubMed Scopus (131) Google Scholar, 3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 4Drager R.G. Higgs D.C. Kindle K.L. Stern D.B. Plant J. 1999; 19: 521-531Crossref PubMed Scopus (56) Google Scholar, 5Fargo D.C. Boynton J.E. Gillham N.W. Plant Cell. 2001; 13: 207-218Crossref PubMed Scopus (22) Google Scholar, 6Hirose T. Sugiura M. EMBO J. 1996; 15: 1687-1695Crossref PubMed Scopus (128) Google Scholar, 7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar, 8McCormac D.J. Litz H. Wang J. Gollnick P.D. Berry J.O. J. Biol. Chem. 2001; 276: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 9Ossenbuhl F. Nickelsen J. Mol. Cell. Biol. 2000; 20: 8134-8142Crossref PubMed Scopus (41) Google Scholar, 10Vaistij F.E. Goldschmidt-Clermont M. Wostrikoff K. Rochaix J.D. Plant J. 2000; 21: 469-482Crossref PubMed Google Scholar). It has been suggested that ribosome association at the 5′-UTR may couple translation with mRNA stability (11Bruick R.K. Mayfield S.P. Nature. 1999; 4: 190-195Google Scholar, 12Mayfield S.P. Yohn C.B. Cohn A. Danon A. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1995; 46: 147-166Crossref Scopus (157) Google Scholar). Most chloroplast mRNAs have an AU-rich 3′-UTR with a terminal inverted repeat. This 3′-UTR inverted repeat has been shown to play a role in the processing and stabilization of the mRNA (13Hayes R. Kudla J. Schuster G. Gabay L. Maliga P. Gruissem W. EMBO J. 1996; 15: 1132-1141Crossref PubMed Scopus (144) Google Scholar, 14Lisitsky I. Klaff P. Schuster G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13398-13403Crossref PubMed Scopus (123) Google Scholar, 15Memon A.R. Meng B. Mullet J.E. Plant Mol. Biol. 1996; 30: 1195-1205Crossref PubMed Scopus (28) Google Scholar, 16Monde R.A. Schuster G. Stern D.B. Biochimie (Paris). 2000; 82: 573-582Crossref PubMed Scopus (113) Google Scholar, 17Schuster G. Gruissem W. EMBO J. 1991; 10: 1493-14502Crossref PubMed Scopus (152) Google Scholar). Identification of interactions between the two termini of cytoplasmic transcripts (18Gallie D.R. Gene (Amst.). 1998; 216: 1-11Crossref PubMed Scopus (239) Google Scholar, 19Mathews M.B. Sonenberg N. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 1-29Google Scholar) and examples of 3′-UTR modulation of translation initiation in both eukaryotes (18Gallie D.R. Gene (Amst.). 1998; 216: 1-11Crossref PubMed Scopus (239) Google Scholar, 20Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1741) Google Scholar) and prokaryotes (21Franch T. Gerdes K. Mol. Microbiol. 1996; 21: 1049-1060Crossref PubMed Scopus (51) Google Scholar, 22Lindahl L. Hinnebusch A. Curr. Opin. Genet. Dev. 1992; 2: 720-726Crossref PubMed Scopus (18) Google Scholar, 23Voorma H.O. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 759-777Google Scholar) raise the possibility that interactions of the 5′- and 3′-UTR of chloroplast mRNAs may influence their expression. In support of this notion, recent results have shown that correct processing of the 3′-UTR can promote translation initiation and polysomal association (24Rott R. Levy H. Drager R.G. Stern D.B. Schuster G. Mol. Cell. Biol. 1998; 18: 4605-4611Crossref PubMed Scopus (39) Google Scholar).Expression of the D1 protein, encoded by the chloroplast psbA mRNA, is a prime example of the regulation of translation involving the interaction of nuclear encoded factors and regions of inverted repeats in the 5′-UTR of the transcript (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 6Hirose T. Sugiura M. EMBO J. 1996; 15: 1687-1695Crossref PubMed Scopus (128) Google Scholar,25Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (132) Google Scholar, 26Girard-Bascou J. Pierre Y. Drapier D. Curr. Genet. 1992; 22: 47-52Crossref PubMed Scopus (55) Google Scholar, 27Staub J.M. Maliga P. Plant J. 1994; 6: 547-553Crossref PubMed Scopus (132) Google Scholar). In C. reinhardtii, the 5′-UTR of psbA mRNA is the target for the light-regulated binding of a complex of proteins of 38, 47, 55, and 60 kDa (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). These proteins interact with an inverted repeat that is located upstream from a potential Shine-Dalgarno-like site in the 5′-UTR (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar) and possibly with a second site after psbA mRNA undergoes 5′-processing (28Bruick R.K. Mayfield S.P. J. Cell Biol. 1998; 143: 1145-1153Crossref PubMed Scopus (47) Google Scholar). Assays of D1 synthesis in C. reinhardtii mutants, in which the inverted repeat has been partially deleted or mutated, indicate the importance of the stem-loop fold for the light-responsive increase of D1 synthesis (7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar).The central RNA-binding protein in the psbA 5′-binding complex is the 47-kDa protein (RB47). RB47 is a nuclear encoded protein that shows high homology to poly(A)-binding proteins (29Yohn C.B. Cohen A. Danon A. Mayfield S.P. Mol. Cell. Biol. 1996; 16: 3560-3566Crossref PubMed Scopus (62) Google Scholar). Mutants in which RB47 expression is low or lacking are defective in D1 translation, implicating RB47 as a message-specific translational factor (29Yohn C.B. Cohen A. Danon A. Mayfield S.P. Mol. Cell. Biol. 1996; 16: 3560-3566Crossref PubMed Scopus (62) Google Scholar, 30Yohn C.B. Cohen A. Rosch C. Kuchka M.R. Mayfield S.P. J. Cell Biol. 1998; 142: 435-442Crossref PubMed Scopus (55) Google Scholar, 31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). The 60-kDa protein (RB60) is a protein-disulfide isomerase-like protein also encoded by the nuclear genome (32Kim J. Mayfield S.P. Science. 1997; 278: 1954-1957Crossref PubMed Scopus (198) Google Scholar). RB60 has been identified as the regulatory redox-active protein of the psbA 5′-binding complex and is the likely candidate for perceiving the light-signal and modulating the binding of the complex (33Trebitsh T. Levitan A. Sofer A. Danon A. Mol. Cell. Biol. 2000; 20: 1116-1123Crossref PubMed Scopus (105) Google Scholar).In addressing the potential modes of regulation of psbA expression, we examined whether the 3′-UTR affects protein binding to psbA mRNA. We report here a comparison of binding of C. reinhardtii proteins to RNA probes of 5′-UTR, 3′-UTR, and cis-linked 5′-UTR-3′-UTR. Our analysis indicated that the high affinity binding to psbA mRNA is primarily via its 5′-UTR, whereas the presence of 3′-UTR in cis increases the affinity of binding of the 5′-UTR-binding protein complex.DISCUSSIONHere we show that the presence in cis of the 3′-UTR of psbA mRNA is required for high affinity binding of the native complex, containing the RB47 protein, to the 5′-UTR. Several modes of action could explain the effect of the 3′-UTR on the binding of the proteins to the 5′-UTR of psbA mRNA. A change in the affinity of an RNA-protein complex may result from a conformational shift in the RNA and/or from an altered binding of the protein. Our results, so far, do not exclude either possibility. The 3′-UTR of psbA mRNA may interact directly with the 5′-UTR of the message, thereby inducing it to form a structural motif with high affinity to the native complex containing RB47. Such long range RNA-RNA interactions have been found to affect translational efficiency in prokaryotes (22Lindahl L. Hinnebusch A. Curr. Opin. Genet. Dev. 1992; 2: 720-726Crossref PubMed Scopus (18) Google Scholar). It should be noted that the MFOLD computer program for RNA structure analysis (36Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar) did not predict any such 5′-UTR-3′-UTR interactions (data not shown). Alternatively, because low affinity binding of RB47 to the 3′-UTR was also found (Fig. 3), it is possible that RB47 may bind both UTRs of psbA mRNA when they are present in cis. This may not be entirely surprising as the 3′-UTR is AU-rich, and RB47 shares high homology with poly(A)-binding proteins (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). In this scheme, the simultaneous binding of RB47 and its associated proteins to the 5′- and 3′-UTRs forms a high affinity complex (Fig. 2).Previous characterization of the 5′-UTR binding activity by GMS, UVCL, and psbA RNA-affinity chromatography assays suggested that it is composed of four proteins (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). The UVCL assay, which labels proteins that are in direct contact with the RNA, showed that RB47 is the primary RNA-binding protein of the complex (Fig. 3 and Ref. 3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar), and cloning of RB47 showed that it contains RNA-binding domains (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). Extraction of the proteins that were complexed with the 5′-UTR of psbA mRNA in the GMS assay revealed that RB60 is also associated with the RNA, potentially via protein-protein interactions with RB47 (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Based on these results, it was suggested that the complex contains four proteins of which only binding of RB47 and RB60 was resistant to the conditions of the GMS (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar).A comparison of mobility shift of 5′-T and 5′3′-T RNAs by either rRB47 or by the partially purified C. reinhardtii proteins showed that the native proteins form complexes with slower mobility (Fig. 6), further indicating that the native complex contains additional protein(s), such as RB60, RB38, or RB55. The higher affinity of the binding of the native complex to the cis-linked 5′- and 3′-UTRs than to the 5′-T transcript indicates that the presence in cis of the 3′-UTR promotes the binding of the native complex comprised of RB47 and its cognate proteins. Interestingly, the recombinant RB47 binds the 5′-T and the 5′3′-T at similar high affinities (compare Figs. 5 and 6), suggesting that the lower affinity of the native complex in the absence of the 3′-UTR is a result of action of the proteins associated with RB47.The activity of the proteins that bind the 5′-UTR psbA mRNA correlates with the translation rate of the D1 protein in C. reinhardtii cells (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Therefore, the increased affinity of 5′-UTR psbA mRNA-binding proteins conferred by the 3′-UTR may suggest that the 3′-UTR influences D1 synthesis by stimulating binding of the native complex, comprised of RB47 and its cognate proteins, to the 5′-UTR. Correct processing of the 3′-UTR was suggested to be required for high levels of translation initiation and polysomal association in C. reinhardtii cells (24Rott R. Levy H. Drager R.G. Stern D.B. Schuster G. Mol. Cell. Biol. 1998; 18: 4605-4611Crossref PubMed Scopus (39) Google Scholar). Recent results from tobacco transformants in which the influence of the psbA UTRs on translation of a reporter gene were studied indicated that including the psbA 3′-UTR resulted in a 3–4-fold enhancement of translation (25Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (132) Google Scholar). In another study, deletion of the inverted repeat of the 3′-UTR of petD mRNA led to a reduction in petD expression beyond that expected by the decrease in mRNA accumulation alone, indicating that the 3′-UTR may also contribute to efficient translation (37Monde R.A. Greene J.C. Stern D.B. Plant Mol. Biol. 2000; 44: 529-542Crossref PubMed Scopus (52) Google Scholar). These findings suggest that the 3′-UTR of chloroplast messages is required for optimal translation, in addition to its role in determination of mRNA stability. Collectively, these results suggest that the impaired binding of the native complex, comprised of RB47 and its associated proteins, to a psbA mRNA lacking an intact 3′-UTR may discriminate it from efficient translation. In initiation of translation, ribosomes bind to the 5′-untranslated region (UTR) 1The abbreviations used are: UTRuntranslated regionGMSgel mobility shiftUVCLUV-cross-linkingrrecombinant 1The abbreviations used are: UTRuntranslated regionGMSgel mobility shiftUVCLUV-cross-linkingrrecombinant of the mRNA and are consequently directed to the initiator codon of the open reading frame. The 5′-UTR of several chloroplast mRNAs in higher plants and in the unicellular green alga Chlamydomonas reinhardtii contain regions of inverted repeats and binding sites for nuclear encoded proteins. These interactions have been shown to be involved not only in regulation of translation but also in processing and stability of the message (1Alexander C. Faber N. Klaff P. Nucleic Acids Res. 1998; 26: 2265-2272Crossref PubMed Scopus (44) Google Scholar, 2Boudreau E. Nickelsen J. Lemaire S.D. Ossenbuhl F. Rochaix J.D. EMBO J. 2000; 19: 3366-3376Crossref PubMed Scopus (131) Google Scholar, 3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 4Drager R.G. Higgs D.C. Kindle K.L. Stern D.B. Plant J. 1999; 19: 521-531Crossref PubMed Scopus (56) Google Scholar, 5Fargo D.C. Boynton J.E. Gillham N.W. Plant Cell. 2001; 13: 207-218Crossref PubMed Scopus (22) Google Scholar, 6Hirose T. Sugiura M. EMBO J. 1996; 15: 1687-1695Crossref PubMed Scopus (128) Google Scholar, 7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar, 8McCormac D.J. Litz H. Wang J. Gollnick P.D. Berry J.O. J. Biol. Chem. 2001; 276: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 9Ossenbuhl F. Nickelsen J. Mol. Cell. Biol. 2000; 20: 8134-8142Crossref PubMed Scopus (41) Google Scholar, 10Vaistij F.E. Goldschmidt-Clermont M. Wostrikoff K. Rochaix J.D. Plant J. 2000; 21: 469-482Crossref PubMed Google Scholar). It has been suggested that ribosome association at the 5′-UTR may couple translation with mRNA stability (11Bruick R.K. Mayfield S.P. Nature. 1999; 4: 190-195Google Scholar, 12Mayfield S.P. Yohn C.B. Cohn A. Danon A. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1995; 46: 147-166Crossref Scopus (157) Google Scholar). Most chloroplast mRNAs have an AU-rich 3′-UTR with a terminal inverted repeat. This 3′-UTR inverted repeat has been shown to play a role in the processing and stabilization of the mRNA (13Hayes R. Kudla J. Schuster G. Gabay L. Maliga P. Gruissem W. EMBO J. 1996; 15: 1132-1141Crossref PubMed Scopus (144) Google Scholar, 14Lisitsky I. Klaff P. Schuster G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13398-13403Crossref PubMed Scopus (123) Google Scholar, 15Memon A.R. Meng B. Mullet J.E. Plant Mol. Biol. 1996; 30: 1195-1205Crossref PubMed Scopus (28) Google Scholar, 16Monde R.A. Schuster G. Stern D.B. Biochimie (Paris). 2000; 82: 573-582Crossref PubMed Scopus (113) Google Scholar, 17Schuster G. Gruissem W. EMBO J. 1991; 10: 1493-14502Crossref PubMed Scopus (152) Google Scholar). Identification of interactions between the two termini of cytoplasmic transcripts (18Gallie D.R. Gene (Amst.). 1998; 216: 1-11Crossref PubMed Scopus (239) Google Scholar, 19Mathews M.B. Sonenberg N. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 1-29Google Scholar) and examples of 3′-UTR modulation of translation initiation in both eukaryotes (18Gallie D.R. Gene (Amst.). 1998; 216: 1-11Crossref PubMed Scopus (239) Google Scholar, 20Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1741) Google Scholar) and prokaryotes (21Franch T. Gerdes K. Mol. Microbiol. 1996; 21: 1049-1060Crossref PubMed Scopus (51) Google Scholar, 22Lindahl L. Hinnebusch A. Curr. Opin. Genet. Dev. 1992; 2: 720-726Crossref PubMed Scopus (18) Google Scholar, 23Voorma H.O. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 759-777Google Scholar) raise the possibility that interactions of the 5′- and 3′-UTR of chloroplast mRNAs may influence their expression. In support of this notion, recent results have shown that correct processing of the 3′-UTR can promote translation initiation and polysomal association (24Rott R. Levy H. Drager R.G. Stern D.B. Schuster G. Mol. Cell. Biol. 1998; 18: 4605-4611Crossref PubMed Scopus (39) Google Scholar). untranslated region gel mobility shift UV-cross-linking recombinant untranslated region gel mobility shift UV-cross-linking recombinant Expression of the D1 protein, encoded by the chloroplast psbA mRNA, is a prime example of the regulation of translation involving the interaction of nuclear encoded factors and regions of inverted repeats in the 5′-UTR of the transcript (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 6Hirose T. Sugiura M. EMBO J. 1996; 15: 1687-1695Crossref PubMed Scopus (128) Google Scholar,25Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (132) Google Scholar, 26Girard-Bascou J. Pierre Y. Drapier D. Curr. Genet. 1992; 22: 47-52Crossref PubMed Scopus (55) Google Scholar, 27Staub J.M. Maliga P. Plant J. 1994; 6: 547-553Crossref PubMed Scopus (132) Google Scholar). In C. reinhardtii, the 5′-UTR of psbA mRNA is the target for the light-regulated binding of a complex of proteins of 38, 47, 55, and 60 kDa (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). These proteins interact with an inverted repeat that is located upstream from a potential Shine-Dalgarno-like site in the 5′-UTR (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar) and possibly with a second site after psbA mRNA undergoes 5′-processing (28Bruick R.K. Mayfield S.P. J. Cell Biol. 1998; 143: 1145-1153Crossref PubMed Scopus (47) Google Scholar). Assays of D1 synthesis in C. reinhardtii mutants, in which the inverted repeat has been partially deleted or mutated, indicate the importance of the stem-loop fold for the light-responsive increase of D1 synthesis (7Mayfield S.P. Cohen A. Danon A. Yohn C.B. J. Cell Biol. 1994; 127: 1537-1545Crossref PubMed Scopus (90) Google Scholar). The central RNA-binding protein in the psbA 5′-binding complex is the 47-kDa protein (RB47). RB47 is a nuclear encoded protein that shows high homology to poly(A)-binding proteins (29Yohn C.B. Cohen A. Danon A. Mayfield S.P. Mol. Cell. Biol. 1996; 16: 3560-3566Crossref PubMed Scopus (62) Google Scholar). Mutants in which RB47 expression is low or lacking are defective in D1 translation, implicating RB47 as a message-specific translational factor (29Yohn C.B. Cohen A. Danon A. Mayfield S.P. Mol. Cell. Biol. 1996; 16: 3560-3566Crossref PubMed Scopus (62) Google Scholar, 30Yohn C.B. Cohen A. Rosch C. Kuchka M.R. Mayfield S.P. J. Cell Biol. 1998; 142: 435-442Crossref PubMed Scopus (55) Google Scholar, 31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). The 60-kDa protein (RB60) is a protein-disulfide isomerase-like protein also encoded by the nuclear genome (32Kim J. Mayfield S.P. Science. 1997; 278: 1954-1957Crossref PubMed Scopus (198) Google Scholar). RB60 has been identified as the regulatory redox-active protein of the psbA 5′-binding complex and is the likely candidate for perceiving the light-signal and modulating the binding of the complex (33Trebitsh T. Levitan A. Sofer A. Danon A. Mol. Cell. Biol. 2000; 20: 1116-1123Crossref PubMed Scopus (105) Google Scholar). In addressing the potential modes of regulation of psbA expression, we examined whether the 3′-UTR affects protein binding to psbA mRNA. We report here a comparison of binding of C. reinhardtii proteins to RNA probes of 5′-UTR, 3′-UTR, and cis-linked 5′-UTR-3′-UTR. Our analysis indicated that the high affinity binding to psbA mRNA is primarily via its 5′-UTR, whereas the presence of 3′-UTR in cis increases the affinity of binding of the 5′-UTR-binding protein complex. DISCUSSIONHere we show that the presence in cis of the 3′-UTR of psbA mRNA is required for high affinity binding of the native complex, containing the RB47 protein, to the 5′-UTR. Several modes of action could explain the effect of the 3′-UTR on the binding of the proteins to the 5′-UTR of psbA mRNA. A change in the affinity of an RNA-protein complex may result from a conformational shift in the RNA and/or from an altered binding of the protein. Our results, so far, do not exclude either possibility. The 3′-UTR of psbA mRNA may interact directly with the 5′-UTR of the message, thereby inducing it to form a structural motif with high affinity to the native complex containing RB47. Such long range RNA-RNA interactions have been found to affect translational efficiency in prokaryotes (22Lindahl L. Hinnebusch A. Curr. Opin. Genet. Dev. 1992; 2: 720-726Crossref PubMed Scopus (18) Google Scholar). It should be noted that the MFOLD computer program for RNA structure analysis (36Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar) did not predict any such 5′-UTR-3′-UTR interactions (data not shown). Alternatively, because low affinity binding of RB47 to the 3′-UTR was also found (Fig. 3), it is possible that RB47 may bind both UTRs of psbA mRNA when they are present in cis. This may not be entirely surprising as the 3′-UTR is AU-rich, and RB47 shares high homology with poly(A)-binding proteins (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). In this scheme, the simultaneous binding of RB47 and its associated proteins to the 5′- and 3′-UTRs forms a high affinity complex (Fig. 2).Previous characterization of the 5′-UTR binding activity by GMS, UVCL, and psbA RNA-affinity chromatography assays suggested that it is composed of four proteins (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). The UVCL assay, which labels proteins that are in direct contact with the RNA, showed that RB47 is the primary RNA-binding protein of the complex (Fig. 3 and Ref. 3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar), and cloning of RB47 showed that it contains RNA-binding domains (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). Extraction of the proteins that were complexed with the 5′-UTR of psbA mRNA in the GMS assay revealed that RB60 is also associated with the RNA, potentially via protein-protein interactions with RB47 (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Based on these results, it was suggested that the complex contains four proteins of which only binding of RB47 and RB60 was resistant to the conditions of the GMS (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar).A comparison of mobility shift of 5′-T and 5′3′-T RNAs by either rRB47 or by the partially purified C. reinhardtii proteins showed that the native proteins form complexes with slower mobility (Fig. 6), further indicating that the native complex contains additional protein(s), such as RB60, RB38, or RB55. The higher affinity of the binding of the native complex to the cis-linked 5′- and 3′-UTRs than to the 5′-T transcript indicates that the presence in cis of the 3′-UTR promotes the binding of the native complex comprised of RB47 and its cognate proteins. Interestingly, the recombinant RB47 binds the 5′-T and the 5′3′-T at similar high affinities (compare Figs. 5 and 6), suggesting that the lower affinity of the native complex in the absence of the 3′-UTR is a result of action of the proteins associated with RB47.The activity of the proteins that bind the 5′-UTR psbA mRNA correlates with the translation rate of the D1 protein in C. reinhardtii cells (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Therefore, the increased affinity of 5′-UTR psbA mRNA-binding proteins conferred by the 3′-UTR may suggest that the 3′-UTR influences D1 synthesis by stimulating binding of the native complex, comprised of RB47 and its cognate proteins, to the 5′-UTR. Correct processing of the 3′-UTR was suggested to be required for high levels of translation initiation and polysomal association in C. reinhardtii cells (24Rott R. Levy H. Drager R.G. Stern D.B. Schuster G. Mol. Cell. Biol. 1998; 18: 4605-4611Crossref PubMed Scopus (39) Google Scholar). Recent results from tobacco transformants in which the influence of the psbA UTRs on translation of a reporter gene were studied indicated that including the psbA 3′-UTR resulted in a 3–4-fold enhancement of translation (25Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (132) Google Scholar). In another study, deletion of the inverted repeat of the 3′-UTR of petD mRNA led to a reduction in petD expression beyond that expected by the decrease in mRNA accumulation alone, indicating that the 3′-UTR may also contribute to efficient translation (37Monde R.A. Greene J.C. Stern D.B. Plant Mol. Biol. 2000; 44: 529-542Crossref PubMed Scopus (52) Google Scholar). These findings suggest that the 3′-UTR of chloroplast messages is required for optimal translation, in addition to its role in determination of mRNA stability. Collectively, these results suggest that the impaired binding of the native complex, comprised of RB47 and its associated proteins, to a psbA mRNA lacking an intact 3′-UTR may discriminate it from efficient translation. Here we show that the presence in cis of the 3′-UTR of psbA mRNA is required for high affinity binding of the native complex, containing the RB47 protein, to the 5′-UTR. Several modes of action could explain the effect of the 3′-UTR on the binding of the proteins to the 5′-UTR of psbA mRNA. A change in the affinity of an RNA-protein complex may result from a conformational shift in the RNA and/or from an altered binding of the protein. Our results, so far, do not exclude either possibility. The 3′-UTR of psbA mRNA may interact directly with the 5′-UTR of the message, thereby inducing it to form a structural motif with high affinity to the native complex containing RB47. Such long range RNA-RNA interactions have been found to affect translational efficiency in prokaryotes (22Lindahl L. Hinnebusch A. Curr. Opin. Genet. Dev. 1992; 2: 720-726Crossref PubMed Scopus (18) Google Scholar). It should be noted that the MFOLD computer program for RNA structure analysis (36Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar) did not predict any such 5′-UTR-3′-UTR interactions (data not shown). Alternatively, because low affinity binding of RB47 to the 3′-UTR was also found (Fig. 3), it is possible that RB47 may bind both UTRs of psbA mRNA when they are present in cis. This may not be entirely surprising as the 3′-UTR is AU-rich, and RB47 shares high homology with poly(A)-binding proteins (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). In this scheme, the simultaneous binding of RB47 and its associated proteins to the 5′- and 3′-UTRs forms a high affinity complex (Fig. 2). Previous characterization of the 5′-UTR binding activity by GMS, UVCL, and psbA RNA-affinity chromatography assays suggested that it is composed of four proteins (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). The UVCL assay, which labels proteins that are in direct contact with the RNA, showed that RB47 is the primary RNA-binding protein of the complex (Fig. 3 and Ref. 3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar), and cloning of RB47 showed that it contains RNA-binding domains (31Yohn C.B. Cohen A. Danon A. Mayfield S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar). Extraction of the proteins that were complexed with the 5′-UTR of psbA mRNA in the GMS assay revealed that RB60 is also associated with the RNA, potentially via protein-protein interactions with RB47 (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Based on these results, it was suggested that the complex contains four proteins of which only binding of RB47 and RB60 was resistant to the conditions of the GMS (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). A comparison of mobility shift of 5′-T and 5′3′-T RNAs by either rRB47 or by the partially purified C. reinhardtii proteins showed that the native proteins form complexes with slower mobility (Fig. 6), further indicating that the native complex contains additional protein(s), such as RB60, RB38, or RB55. The higher affinity of the binding of the native complex to the cis-linked 5′- and 3′-UTRs than to the 5′-T transcript indicates that the presence in cis of the 3′-UTR promotes the binding of the native complex comprised of RB47 and its cognate proteins. Interestingly, the recombinant RB47 binds the 5′-T and the 5′3′-T at similar high affinities (compare Figs. 5 and 6), suggesting that the lower affinity of the native complex in the absence of the 3′-UTR is a result of action of the proteins associated with RB47. The activity of the proteins that bind the 5′-UTR psbA mRNA correlates with the translation rate of the D1 protein in C. reinhardtii cells (3Danon A. Mayfield S.P. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar). Therefore, the increased affinity of 5′-UTR psbA mRNA-binding proteins conferred by the 3′-UTR may suggest that the 3′-UTR influences D1 synthesis by stimulating binding of the native complex, comprised of RB47 and its cognate proteins, to the 5′-UTR. Correct processing of the 3′-UTR was suggested to be required for high levels of translation initiation and polysomal association in C. reinhardtii cells (24Rott R. Levy H. Drager R.G. Stern D.B. Schuster G. Mol. Cell. Biol. 1998; 18: 4605-4611Crossref PubMed Scopus (39) Google Scholar). Recent results from tobacco transformants in which the influence of the psbA UTRs on translation of a reporter gene were studied indicated that including the psbA 3′-UTR resulted in a 3–4-fold enhancement of translation (25Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (132) Google Scholar). In another study, deletion of the inverted repeat of the 3′-UTR of petD mRNA led to a reduction in petD expression beyond that expected by the decrease in mRNA accumulation alone, indicating that the 3′-UTR may also contribute to efficient translation (37Monde R.A. Greene J.C. Stern D.B. Plant Mol. Biol. 2000; 44: 529-542Crossref PubMed Scopus (52) Google Scholar). These findings suggest that the 3′-UTR of chloroplast messages is required for optimal translation, in addition to its role in determination of mRNA stability. Collectively, these results suggest that the impaired binding of the native complex, comprised of RB47 and its associated proteins, to a psbA mRNA lacking an intact 3′-UTR may discriminate it from efficient translation.

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