Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1990 Oct;9(10):3059–3066. doi: 10.1002/j.1460-2075.1990.tb07502.x

Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs.

Y Q Li 1, M Sugiura 1
PMCID: PMC552030  PMID: 1698606

Abstract

Chloroplasts contain their own genetic system. Eighteen different split genes have been found among approximately 130 chloroplast genes from higher plants. However, little is known about the chloroplast splicing system. Mammalian heterogeneous nuclear ribonucleoproteins (hnRNP proteins) have been shown to be involved in splicing. We applied a purification procedure developed for HeLa cell hnRNP proteins, which uses a single-stranded DNA (ssDNA) affinity column, directly to the tobacco chloroplast lysate to isolate their chloroplast counterparts. Four proteins (mol. wt approximately 30 kd) bound strongly to the column. The amino-terminal sequences of three of them were determined and their cDNA clones were isolated from a tobacco leaf cDNA library. Sequence analysis of these clones revealed that all three proteins contain two ribonucleoprotein consensus sequences (RNP-CS), confirming their ribonucleoprotein (RNP) nature. The presence of putative transit peptides in their predicted protein sequences, and an in vitro import experiment confirmed they are located in the chloroplast. This is the first report of organellar proteins containing RNP-CS. In addition, these three chloroplast proteins have a very acidic amino-terminal domain, a novel feature among RNP proteins identified so far. They are expressed both in leaves and roots; their mRNA levels showed different light modulation in mature leaves. The three proteins might be involved in splicing and/or processing of chloroplast RNAs.

Full text

PDF
3059

Images in this article

Selected References

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

  1. Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson G. J., Bach M., Lührmann R., Beggs J. D. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein. Nature. 1989 Dec 14;342(6251):819–821. doi: 10.1038/342819a0. [DOI] [PubMed] [Google Scholar]
  3. Angelini G., de Preval C., Gorski J., Mach B. High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4489–4493. doi: 10.1073/pnas.83.12.4489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  5. Bourbon H. M., Lapeyre B., Amalric F. Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons. J Mol Biol. 1988 Apr 20;200(4):627–638. doi: 10.1016/0022-2836(88)90476-7. [DOI] [PubMed] [Google Scholar]
  6. Burd C. G., Swanson M. S., Görlach M., Dreyfuss G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9788–9792. doi: 10.1073/pnas.86.24.9788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buvoli M., Biamonti G., Tsoulfas P., Bassi M. T., Ghetti A., Riva S., Morandi C. cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms. Nucleic Acids Res. 1988 May 11;16(9):3751–3770. doi: 10.1093/nar/16.9.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
  9. Dreyfuss G., Swanson M. S., Piñol-Roma S. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci. 1988 Mar;13(3):86–91. doi: 10.1016/0968-0004(88)90046-1. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  11. Gómez J., Sánchez-Martínez D., Stiefel V., Rigau J., Puigdomènech P., Pagès M. A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein. Nature. 1988 Jul 21;334(6179):262–264. doi: 10.1038/334262a0. [DOI] [PubMed] [Google Scholar]
  12. Hiratsuka J., Shimada H., Whittier R., Ishibashi T., Sakamoto M., Mori M., Kondo C., Honji Y., Sun C. R., Meng B. Y. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet. 1989 Jun;217(2-3):185–194. doi: 10.1007/BF02464880. [DOI] [PubMed] [Google Scholar]
  13. Kück U., Choquet Y., Schneider M., Dron M., Bennoun P. Structural and transcription analysis of two homologous genes for the P700 chlorophyll a-apoproteins in Chlamydomonas reinhardii: evidence for in vivo trans-splicing. EMBO J. 1987 Aug;6(8):2185–2195. doi: 10.1002/j.1460-2075.1987.tb02489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Manfioletti G., Schneider C. A new and fast method for preparing high quality lambda DNA suitable for sequencing. Nucleic Acids Res. 1988 Apr 11;16(7):2873–2884. doi: 10.1093/nar/16.7.2873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marcu K., Dudock B. Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucleic Acids Res. 1974 Nov;1(11):1385–1397. doi: 10.1093/nar/1.11.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  18. Mattaj I. W. A binding consensus: RNA-protein interactions in splicing, snRNPs, and sex. Cell. 1989 Apr 7;57(1):1–3. doi: 10.1016/0092-8674(89)90164-5. [DOI] [PubMed] [Google Scholar]
  19. Merrill B. M., Stone K. L., Cobianchi F., Wilson S. H., Williams K. R. Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein. J Biol Chem. 1988 Mar 5;263(7):3307–3313. [PubMed] [Google Scholar]
  20. Minami E., Shinohara K., Kuwabara T., Watanabe A. In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts. Arch Biochem Biophys. 1986 Feb 1;244(2):517–527. doi: 10.1016/0003-9861(86)90620-x. [DOI] [PubMed] [Google Scholar]
  21. Mortenson E., Dreyfuss G. RNP in maize protein. Nature. 1989 Jan 26;337(6205):312–312. doi: 10.1038/337312b0. [DOI] [PubMed] [Google Scholar]
  22. Münch S., Ljungberg U., Steppuhn J., Schneiderbauer A., Nechushtai R., Beyreuther K., Herrmann R. G. Nucleotide sequences of cDNAs encoding the entire precursor polypeptides for subunits II and III of the photosystem I reaction center from spinach. Curr Genet. 1988 Nov;14(5):511–518. doi: 10.1007/BF00521277. [DOI] [PubMed] [Google Scholar]
  23. Obokata J., Mikami K., Hayashida N., Sugiura M. Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana. Plant Physiol. 1990 Jan;92(1):273–275. doi: 10.1104/pp.92.1.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pandolfo M., Valentini O., Biamonti G., Rossi P., Riva S. Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose. Eur J Biochem. 1987 Jan 2;162(1):213–220. doi: 10.1111/j.1432-1033.1987.tb10563.x. [DOI] [PubMed] [Google Scholar]
  25. Pinck M., Guilley E., Durr A., Hoff M., Pinck L., Fleck J. Complete sequence of one of the mRNAs coding for the small subunit of ribulose bisphosphate carboxylase of Nicotiana sylvestris. Biochimie. 1984 Jul-Aug;66(7-8):539–545. doi: 10.1016/0300-9084(84)90148-2. [DOI] [PubMed] [Google Scholar]
  26. Piñol-Roma S., Choi Y. D., Matunis M. J., Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. doi: 10.1101/gad.2.2.215. [DOI] [PubMed] [Google Scholar]
  27. Preugschat F., Wold B. Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9669–9673. doi: 10.1073/pnas.85.24.9669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  29. Robinow S., Campos A. R., Yao K. M., White K. The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs. Science. 1988 Dec 16;242(4885):1570–1572. doi: 10.1126/science.3144044. [DOI] [PubMed] [Google Scholar]
  30. Rock C. D., Barkan A., Taylor W. C. The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products. Curr Genet. 1987;12(1):69–77. doi: 10.1007/BF00420729. [DOI] [PubMed] [Google Scholar]
  31. Sachs A. B., Bond M. W., Kornberg R. D. A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression. Cell. 1986 Jun 20;45(6):827–835. doi: 10.1016/0092-8674(86)90557-x. [DOI] [PubMed] [Google Scholar]
  32. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmidt G. W., Mishkind M. L. The transport of proteins into chloroplasts. Annu Rev Biochem. 1986;55:879–912. doi: 10.1146/annurev.bi.55.070186.004311. [DOI] [PubMed] [Google Scholar]
  34. Shinozaki K., Ohme M., Tanaka M., Wakasugi T., Hayashida N., Matsubayashi T., Zaita N., Chunwongse J., Obokata J., Yamaguchi-Shinozaki K. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986 Sep;5(9):2043–2049. doi: 10.1002/j.1460-2075.1986.tb04464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Skinner M. K., Griswold M. D. Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem J. 1983 Jan 1;209(1):281–284. doi: 10.1042/bj2090281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sugita M., Gruissem W. Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7104–7108. doi: 10.1073/pnas.84.20.7104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sugiura M. The chloroplast chromosomes in land plants. Annu Rev Cell Biol. 1989;5:51–70. doi: 10.1146/annurev.cb.05.110189.000411. [DOI] [PubMed] [Google Scholar]
  38. Surowy C. S., van Santen V. L., Scheib-Wixted S. M., Spritz R. A. Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA. Mol Cell Biol. 1989 Oct;9(10):4179–4186. doi: 10.1128/mcb.9.10.4179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swanson M. S., Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. doi: 10.1128/mcb.8.5.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Swanson M. S., Nakagawa T. Y., LeVan K., Dreyfuss G. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. Mol Cell Biol. 1987 May;7(5):1731–1739. doi: 10.1128/mcb.7.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tanaka M, Obokata J, Chunwongse J, Shinozaki K, Sugiura M. Rapid splicing and stepwise processing of a transcript from the psbB operon in tobacco chloroplasts: determination of the intron sites in petB and petD. Mol Gen Genet. 1987 Oct;209(3):427–431. doi: 10.1007/BF00331145. [DOI] [PubMed] [Google Scholar]
  43. Wallace R. B., Miyada C. G. Oligonucleotide probes for the screening of recombinant DNA libraries. Methods Enzymol. 1987;152:432–442. doi: 10.1016/0076-6879(87)52050-x. [DOI] [PubMed] [Google Scholar]
  44. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  45. Westhoff P., Farchaus J. W., Herrmann R. G. The gene for the Mr 10,000 phosphoprotein associated with photosystem II is part of the psbB operon of the spinach plastid chromosome. Curr Genet. 1986;11(3):165–169. doi: 10.1007/BF00420602. [DOI] [PubMed] [Google Scholar]
  46. Westhoff P., Herrmann R. G. Complex RNA maturation in chloroplasts. The psbB operon from spinach. Eur J Biochem. 1988 Feb 1;171(3):551–564. doi: 10.1111/j.1432-1033.1988.tb13824.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES