Plant Molecular Biology

, Volume 30, Issue 1, pp 109–123

Impaired splicing of the rps 12 transcript in ribosome-deficient plastids

  • Thomas Hübschmann
  • Wolfgang R. Hess
  • Thomas Börner
Research Article


Analysis of RNA maturation in ribosome-deficient plastids of four non-allelic barley mutants revealed an increased accumulation and altered processing of transcripts of the ribosomal protein gene CS12 (rps12) compared to normal chloroplasts. The three exons of rps12 are part of two different polycistronic transcription units. Generation of mature rps12-mRNA involves both cis-and trans-splicing. In ribosome-deficient plastids, the cis-intron separating exons 2 and 3 remains entirely unspliced whereas the splicing of the bipartite rps12 trans-intron between exon 1 and exon 2 occurs, but at a reduced level. A comparison of the 3′ and 5′ ends of the two RNAs that are generally assumed to interact during trans-splicing showed a difference in the processing pathways of 3′ rps12 transcripts between mutated and normal chloroplasts. Nonetheless, the final products were identical.

Key words

group II intron Hordeum vulgare RNA processing rps 12 splicing transcription 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Adams CC, Stern DB: Control of mRNA stability in chloroplasts by 3′ inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro. Nucl Acids Res 18: 6003–6010 (1990).Google Scholar
  2. 2.
    Apirion D, Miczak A: RNA processing in procaryotic cells. Bioessays 15: 113–120 (1993).Google Scholar
  3. 3.
    Arnason TJ, Walker GWR: An irreversible gene-induced plastid mutation. Can J Res 27C: 172–178 (1949).Google Scholar
  4. 4.
    Attardi G, Schatz G: Biogenesis of mitochondria. Annu Rev Cell Biol 4: 289–333 (1988).Google Scholar
  5. 5.
    Banta AB, Haas ES, Brown JW, Pace NR: Sequence of the ribonuclease P RNA gene from the cyanobacterium Anacystis nidulans. Nucl Acids Res 20: 911 (1992).Google Scholar
  6. 6.
    Barkan A, Walker M, Nolasco M, Johnson D: A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms. EMBO J 13: 3170–3181 (1994).Google Scholar
  7. 7.
    Barkan A: Nuclear mutants of maize with defects in chloroplast polysome assembly have altered chloroplast RNA metabolism. Plant Cell 5: 389–402 (1993).Google Scholar
  8. 8.
    Barkan A: Proteins encoded by a complex chloroplast transcription unit are translated from both monocistronic and polycistronic mRNAs. EMBO J 7: 2637–2644 (1988).Google Scholar
  9. 9.
    Barkan A, Miles D, Taylor WC: Chloroplast gene expression in nuclear, photosynthetic mutants of maize. EMBO J 5: 1421–1427 (1985).Google Scholar
  10. 10.
    Börner T, Sears B: Plastome mutants. Plant Mol Biol Rep 4: 68–92 (1985).Google Scholar
  11. 11.
    Börner T, Schumann B, Hagemann R: Biochemical studies on a plastid ribosome-deficient mutant of Hordeum vulgare. In: Bücher T, Neupert W, Sebald W, Werner S (eds) Genetic and Biogenesis of Chloroplasts and Mitochondria, p pp. 41–48. Elsevier/North Holland, Amsterdam (1976).Google Scholar
  12. 12.
    Coetzee T, Herschlag D, Belfort M: Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones. Genes Devel 8: 1575–1588 (1994).Google Scholar
  13. 13.
    Deutscher MP: Ribonuclease multiplicity, diversity, and complexity. J Biol Chem 268: 13011–13014 (1993).Google Scholar
  14. 14.
    Ems SC, Morden CW, Dixon CK, Wolfe KH, dePamphilis CW, Palmer JD: Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana. Plant Mol Biol, in press.Google Scholar
  15. 15.
    Falk J, Schmidt A, Krupinska K: Characterization of plastid DNA transcription in ribosome deficient plastids of heat-bleached barley leaves. J Plant Physiol 141: 176–181 (1993).Google Scholar
  16. 16.
    Godefroy-Colburn T, Grunberg-Manago M: Polynucleotide phosphorylase. In: Boyer PD (ed) The Enzymes, vol. 7, pp. 533–574, Academic Press, New York (1972).Google Scholar
  17. 17.
    Goldschmidt-Clermont M, Choquet Y, Girard-Bascou J, Michel F, Schirmer-Rahire M, Rochaix JD: A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii. Cell 65: 135–143 (1990).Google Scholar
  18. 18.
    Goldschmidt-Clermont M, Girard-Bascou J, Choquet Y, Rochaix JD: Trans-splicing mutants of Chlamydomonas reinhardtii. Mol Gen Genet 223: 417–425 (1990).Google Scholar
  19. 19.
    Gruissem W, Schuster G: Control of mRNA degradation in organelles. In: Belasco JG, Brawerman G (eds) Control of Messenger RNA Stability, pp. 329–365. Academic Press, New York (1993).Google Scholar
  20. 20.
    Gruissem W, Tonkyn JC: Control mechanisms of plastid gene expression. Crit Rev Plant Sci 10: 525–558 (1993).Google Scholar
  21. 21.
    Hagemann R, Scholz F: Ein Fall Gen-induzierter Mutationen des Plasmotyps bei Gerste. Züchter 32: 50–59 (1962).Google Scholar
  22. 22.
    Henningsen KW, Boynton JE, von Wettstein D: Mutants at xantha and albina loci in relation to chloroplast biogenesis in barley (Hordeum vulgare L.). Copenhagen, Denmark: The Royal Danish Academy of Sciences and Letters. Munksgaard, Copenhagen (1993).Google Scholar
  23. 23.
    Hess WR, Hoch B, Zeltz P, Hübschmann T, Kössel H, Börner T: Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids. Plant Cell 6: 1455–1465 (1994).Google Scholar
  24. 24.
    Hess WR, Müller A, Nagy F, Börner T: Ribosome-deficient plastids affect transcription of light-induced nuclear genes: genetic evidence for a plastid-derived signal. Mol Gen Genet 242: 305–312 (1994).Google Scholar
  25. 25.
    Hess WR, Prombona A, Fieder B, Subramanian AR, Börner T: Chloroplast rps15 and rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase. EMBO J 12: 563–571 (1993).Google Scholar
  26. 26.
    Hess WR, Schendel R, Rüdiger W, Fieder B, Börner T: Components of chlorophyll biosynthesis in a barley albina mutant unable to synthesize δ-aminolevulinic acid by utilizing the transfer RNA for glutamic acid. Planta 188: 19–27 (1992).Google Scholar
  27. 27.
    Hildebrand M, Hallick RB, Passavant CW, Bourque DP: Trans-splicing in chloroplasts: The rps12 loci of Nicotiana tabacum. Proc Natl Acad Sci USA 85: 372–376 (1986).Google Scholar
  28. 28.
    Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun CR, Meng BY, Li YQ, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M: The complete sequence of 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 217: 185–194 (1989).Google Scholar
  29. 29.
    Iratni R, Baeza L, Andreeva A, Mache R, Lerbs-Mache S: Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression. Genes Devel 8: 2928–2938 (1994).Google Scholar
  30. 30.
    Kanno A, Hirai A: A transcription map of the chloroplast genome from rice (Oryza sativa). Curr Genet 23: 166–174 (1993).Google Scholar
  31. 31.
    Kohchi T, Umesono K, Ogura Y, Komine Y, Nakahigashi K, Komano T, Yamada Y, Ozeki H, Ohyama K: A nicked group II intron and trans-splicing in liverwort, Marchantia polymorpha, chloroplasts. Nucl Acids Res 16: 10025–10036 (1988).Google Scholar
  32. 32.
    Lambowitz AM, Belfort M: Introns as mobile genetic elements. Annu Rev Biochem 62: 587–622 (1993).Google Scholar
  33. 33.
    Liere K, Link G: RNA-binding activity of the matK protein encoded by the chloroplast trnK intron from mustard (Sinapis alba L.) Nucl Acids Res 23: 917–921 (1995).Google Scholar
  34. 34.
    Maurizi MR, William PC, Seung-Ho K, Gottesman S: ClpP represents a unique family of serine proteases. J Biol Chem 265: 12546–12552 (1990).Google Scholar
  35. 35.
    Michel F, Umesono K, Ozeki H: Comparative and functional anatomy of group II catalytic introns: a review. Gene 82: 5–30 (1989).Google Scholar
  36. 36.
    Mohr G, Perlman PS, Lambowitz AM: Evolutionary relationship among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function. Nucl Acids Res 21: 4991–4997 (1993).Google Scholar
  37. 37.
    Moran JV, Mecklenburg KL, Sass P, Belcher SM, Mahnke D, Lewin A, Perlman P: Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron A12. Nucl Acids Res 22: 2057–2064 (1994).Google Scholar
  38. 38.
    Mullet JE: Dynamic regulation of chloroplast transcription. Plant Physiol 103: 309–313 (1994).Google Scholar
  39. 39.
    Nickelsen J, Dillewijn JV, Rahire M, Rochaix JD: Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. EMBO J 13: 3182–3191 (1994).Google Scholar
  40. 40.
    Nickelsen J, Link G: The 54 kDa RNA-binding protein from mustard chloroplasts mediates endonucleolytic transcript 3′ end formation in vitro. Plant J 3: 537–544 (1993).Google Scholar
  41. 41.
    Nilsen TW: RNA-RNA interactions in the spliceosome: unraveling the ties that bind. Cell 78: 1–4 (1994).Google Scholar
  42. 42.
    Ohta M, Sugita M, Sugiura M: Three types of nuclear genes encoding chloroplast RNA-binding proteins (cp29, cp31 and cp33) are present in Arabidopsis thaliana: presence of cp31 in chloroplasts and its homologue in nuclei/cytoplasm. Plant Mol Biol 27: 529–539 (1995).Google Scholar
  43. 43.
    Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H: Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574 (1986).Google Scholar
  44. 44.
    Pace NR, Brown JW: Evolutionary perspective on structure and function of ribonuclease P, a ribozyme. J Bact 17: 1919–1928 (1995).Google Scholar
  45. 45.
    Paulsen H, Bogorad L: Diurnal and circadian rhythm in the accumulation and synthesis of mRNA for the light-harvesting chlorophyll a/b binding protein in tobacco. Plant Physiol 88: 1104–1109 (1988).Google Scholar
  46. 46.
    Post LE, Nomura M: DNA sequences from the str operon of Escherichia coli. J Biol Chem 255: 4660–4666 (1980).Google Scholar
  47. 47.
    Rochaix JD: Post-transcriptional steps in the expression of chloroplast genes. Annu Rev Cell Biol 8: 1–28 (1992).Google Scholar
  48. 48.
    Rogers SO, Bendich AI: Extraction of DNA from milligram amounts of fresh, herbarium, and mummified plant tissues. Plant Mol Biol 5: 69–76 (1985).Google Scholar
  49. 49.
    Saldanha R, Mohr G, Belfort M, Lambowitz AM: Group I and group II introns. FASEB J 7: 15–24 (1993).Google Scholar
  50. 50.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  51. 51.
    Schuster G, Gruissem W: Chloroplast mRNA 3′ end processing requires a nuclear-encoded RNA-binding protein. EMBO J 10: 1493–1502 (1991).Google Scholar
  52. 52.
    Sexton TB, Jones JT, Mullet JE: Sequence and trancriptional analysis of the barley cDNA region upstream of psbD-psbC encoding trnK(UUU), rps16, trnQ(UUG), psbK, psbI and trnS(GCU). Curr Genet 17: 445–454 (1990).Google Scholar
  53. 53.
    Shimada H, Sugiura M: Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes. Nucl Acids Res 19: 983–995 (1991).Google Scholar
  54. 54.
    Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira H, Yamada K, Kusuda J, Takaiwa F, Kato A, Shimada A, Sugiura M: The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043–2049 (1986).Google Scholar
  55. 55.
    Stern DB, Kindle KL: 3′ end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process. Mol Cel Biol 13: 2277–2285 (1993).Google Scholar
  56. 56.
    Stern DB, Gruissem W: Chloroplast mRNA 3′ end maturation is biochemical distinct from prolaryotic mRNA processing. Plant Mol Biol 13: 615–625 (1989).Google Scholar
  57. 57.
    Sugiura M: The chloroplast genome. Plant Mol Biol 19: 149–168 (1992).Google Scholar
  58. 58.
    Turmel M, Choquet Y, Goldschmidt-Clermont M, Rochaix JD, Otis C, Lemieux C: The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis. Curr Genet 27: 270–279 (1995).Google Scholar
  59. 59.
    Vera A, Sugiura M: A novel RNA gene in the tobacco plastid genome: its possible role in the maturation of 16S rRNA. EMBO J 13: 2211–2217 (1994).Google Scholar
  60. 60.
    Wang MJ, Davis NW, Gegenheimer P: Novel mechanisms for maturation of chloroplast transfer RNA precursors. EMBO J: 1567–1574 (1988).Google Scholar
  61. 61.
    Weglöhner W, Subramanian AR: Nucleotide sequence of a region of maize chloroplast DNA containing the 3′ end of clpP, exon 1 of rps12 and rpl20 and their cotranscription. Plant Mol Biol 18: 415–418 (1992).Google Scholar
  62. 62.
    Westhoff P, Herrmann RG: Complex RNA maturation in chloroplasts: the psbB operon from spinach. Eur J Biochem 171: 551–564 (1988).Google Scholar
  63. 63.
    Ye L, Li Y, 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. Nucl Acids Res 19: 6485–6490 (1991).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Thomas Hübschmann
    • 1
  • Wolfgang R. Hess
    • 1
  • Thomas Börner
    • 1
  1. 1.Department of BiologyHumboldt-University BerlinBerlinGermany

Personalised recommendations