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. 1995 Jul 11;23(13):2506–2511. doi: 10.1093/nar/23.13.2506

Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA.

I Lisitsky 1, G Schuster 1
PMCID: PMC307058  PMID: 7630729

Abstract

An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain. Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs. It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs. In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases. It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with [32P] orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II. The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein. Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan. RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein. Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams C. C., Stern D. B. Control of mRNA stability in chloroplasts by 3' inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro. Nucleic Acids Res. 1990 Oct 25;18(20):6003–6010. doi: 10.1093/nar/18.20.6003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Zvi D., Shagan T., Schindler U., Cashmore A. R. RNP-T, a ribonucleoprotein from Arabidopsis thaliana, contains two RNP-80 motifs and a novel acidic repeat arranged in an alpha-helix conformation. Plant Mol Biol. 1992 Dec;20(5):833–838. doi: 10.1007/BF00027154. [DOI] [PubMed] [Google Scholar]
  3. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boldyreff B., Meggio F., Dobrowolska G., Pinna L. A., Issinger O. G. Expression and characterization of a recombinant maize CK-2 alpha subunit. Biochim Biophys Acta. 1993 Apr 29;1173(1):32–38. doi: 10.1016/0167-4781(93)90239-a. [DOI] [PubMed] [Google Scholar]
  5. Burd C. G., Dreyfuss G. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 1994 Mar 1;13(5):1197–1204. doi: 10.1002/j.1460-2075.1994.tb06369.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cobianchi F., Calvio C., Stoppini M., Buvoli M., Riva S. Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):949–955. doi: 10.1093/nar/21.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cook W. B., Walker J. C. Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins. Nucleic Acids Res. 1992 Jan 25;20(2):359–364. doi: 10.1093/nar/20.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Danon A., Mayfield S. P. ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation. EMBO J. 1994 May 1;13(9):2227–2235. doi: 10.1002/j.1460-2075.1994.tb06500.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeLisle A. J. RNA-Binding Protein from Arabidopsis. Plant Physiol. 1993 May;102(1):313–314. doi: 10.1104/pp.102.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  11. Feng L., Yoon H., Donahue T. F. Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae. Mol Cell Biol. 1994 Aug;14(8):5139–5153. doi: 10.1128/mcb.14.8.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukami-Kobayashi K., Tomoda S., Go M. Evolutionary clustering and functional similarity of RNA-binding proteins. FEBS Lett. 1993 Dec 6;335(2):289–293. doi: 10.1016/0014-5793(93)80749-k. [DOI] [PubMed] [Google Scholar]
  13. Gruissem W. Chloroplast gene expression: how plants turn their plastids on. Cell. 1989 Jan 27;56(2):161–170. doi: 10.1016/0092-8674(89)90889-1. [DOI] [PubMed] [Google Scholar]
  14. Gruissem W., Greenberg B. M., Zurawski G., Hallick R. B. Chloroplast gene expression and promoter identification in chloroplast extracts. Methods Enzymol. 1986;118:253–270. doi: 10.1016/0076-6879(86)18077-3. [DOI] [PubMed] [Google Scholar]
  15. Issinger O. G. Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol Ther. 1993;59(1):1–30. doi: 10.1016/0163-7258(93)90039-g. [DOI] [PubMed] [Google Scholar]
  16. Kanekatsu M., Munakata H., Furuzono K., Ohtsuki K. Biochemical characterization of a 34 kDa ribonucleoprotein (p34) purified from the spinach chloroplast fraction as an effective phosphate acceptor for casein kinase II. FEBS Lett. 1993 Dec 6;335(2):176–180. doi: 10.1016/0014-5793(93)80724-9. [DOI] [PubMed] [Google Scholar]
  17. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  18. Klimczak L. J., Schindler U., Cashmore A. R. DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli. Plant Cell. 1992 Jan;4(1):87–98. doi: 10.1105/tpc.4.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Y. Q., Sugiura M. Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs. EMBO J. 1990 Oct;9(10):3059–3066. doi: 10.1002/j.1460-2075.1990.tb07502.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lisitsky I., Liveanu V., Schuster G. RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast. Plant Physiol. 1995 Mar;107(3):933–941. doi: 10.1104/pp.107.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lisitsky I., Liveanu V., Schuster G. RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein. Nucleic Acids Res. 1994 Nov 11;22(22):4719–4724. doi: 10.1093/nar/22.22.4719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lisitsky I., Schuster G. A method to determine the minimal number of nucleotides required for the binding of a ribonucleoprotein to RNA. Anal Biochem. 1995 Jan 20;224(2):603–605. doi: 10.1006/abio.1995.1094. [DOI] [PubMed] [Google Scholar]
  23. Marciniak R. A., Garcia-Blanco M. A., Sharp P. A. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1990 May;87(9):3624–3628. doi: 10.1073/pnas.87.9.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mieszczak M., Klahre U., Levy J. H., Goodall G. J., Filipowicz W. Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia. Mol Gen Genet. 1992 Sep;234(3):390–400. doi: 10.1007/BF00538698. [DOI] [PubMed] [Google Scholar]
  25. Pugh B. F., Tjian R. Mechanism of transcriptional activation by Sp1: evidence for coactivators. Cell. 1990 Jun 29;61(7):1187–1197. doi: 10.1016/0092-8674(90)90683-6. [DOI] [PubMed] [Google Scholar]
  26. Rochaix J. D. Post-transcriptional steps in the expression of chloroplast genes. Annu Rev Cell Biol. 1992;8:1–28. doi: 10.1146/annurev.cb.08.110192.000245. [DOI] [PubMed] [Google Scholar]
  27. Schuster G., Gruissem W. Chloroplast mRNA 3' end processing requires a nuclear-encoded RNA-binding protein. EMBO J. 1991 Jun;10(6):1493–1502. doi: 10.1002/j.1460-2075.1991.tb07669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stern D. B., Gruissem W. Control of plastid gene expression: 3' inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell. 1987 Dec 24;51(6):1145–1157. doi: 10.1016/0092-8674(87)90600-3. [DOI] [PubMed] [Google Scholar]
  29. Stern D. B., Jones H., Gruissem W. Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding. J Biol Chem. 1989 Nov 5;264(31):18742–18750. [PubMed] [Google Scholar]
  30. Stern D. B., Radwanski E. R., Kindle K. L. A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell. 1991 Mar;3(3):285–297. doi: 10.1105/tpc.3.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ye L. H., Li Y. Q., Fukami-Kobayashi K., Go M., Konishi T., Watanabe A., Sugiura M. Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA-binding domains. Nucleic Acids Res. 1991 Dec 11;19(23):6485–6490. doi: 10.1093/nar/19.23.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ye L. H., Li Y. Q., Fukami-Kobayashi K., Go M., Konishi T., Watanabe A., Sugiura M. Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA-binding domains. Nucleic Acids Res. 1991 Dec 11;19(23):6485–6490. doi: 10.1093/nar/19.23.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]

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