Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Action mechanism of upstream open reading frame from S-adenosylmethionine decarboxylase gene as a in vivo translational inhibitor

S-Adenosylmethionine decarboxylase 유전자의 upstream open reading frame이 in vivo에서 translational inhibitor 로서의 작용 기작

  • Choi, Yu-Jin (Department of Biology, Sunchon National University) ;
  • Park, Ky-Young (Department of Biology, Sunchon National University)
  • 최유진 (순천대학교 생명산업과학대학 생물학과) ;
  • 박기영 (순천대학교 생명산업과학대학 생물학과)
  • Received : 2011.03.14
  • Accepted : 2011.03.20
  • Published : 2011.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.

SAMDC는 폴리아민 생합성 과정에서 주효소로 작용하며 항상성을 유지하기위해 정교하게 조절된다. 카네이션 SAMDC 유전자는 5'-leader sequence에 54개 아미노산으로 구성된 small uORF가 존재한다. Translation 과정을 조절하는 uORF의 작용기작을 연구하기 위하여 35S 프로모터에 SAMDC 유전자의 uORF 부위와 GUS 유전자를 재조합한 형질전환 담배 식물체를 이용하였다. 본 실험에서는 SAMDC uORF 염기서열 혹은 SAMDC uORF 단백질에 의해서 downstream GUS ORF의 translation이 억제되었다. 특히 translation 억제는 개시코돈이 point-mutation된 construct에서 효과적으로 이루어졌다. 따라서 이러한 결과는 ribosomal stalling이 translation 억제 과정에 관여한 것으로 사료된다. 개시 코돈과 종결코돈을 가진 SAMDC uORF의 아미노산 서열을 frame shift 시키면 GUS 활성이 증가하였는데 이는 translation inhibitor로서 작용할 때 아미노산 서열이 중요하다는 것을 의미하며, 결국은 SAMDC uORF의 단백질 구조가 중요하게 작용할 가능성을 제시한다. 또한 유식물과 담배 꽃 등의 in vivo 상에서도 GUS 발현을 조직화학적으로 분석했을 때 small uORF가 존재할 경우 GUS 염색이 크게 저하되었지만, 개시코돈이나 혹은 종결코돈이 제거되도록 point-mutation 시킨 construct가 도입된 형질전환식물체에서는 SAMDC uORF의 억제효과가 크게 완화 되었다. 또한 가장 중요한 관찰 결과로는 small uORF 염기서열로부터 in vitro 시스템에서 5.7 kDa의 단백질이 실제적으로 합성되었음을 관찰하였다. 폴리아민 처리 후 GUS 단백질이 억제된 결과는 uORF로부터 합성된 단백질이 폴리아민 뿐 만 아니라 translation 과정에 관여하는 다른 요소들과 상호작용을 이루어 조절될 수 있음을 암시한다.

Keywords

References

  1. Agostinelli E, Bachrach U (2007) Polyamines and their analogs in cancer and other diseases. Amino Acids 33:173 https://doi.org/10.1007/s00726-007-0538-8
  2. Bouchereau A, Aziz A, Larher F, Martin-Tanguy J (1999) Polyamines and environmental challenges: recent development. Plant Sci 140:103-125 https://doi.org/10.1016/S0168-9452(98)00218-0
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Colin H, Marina F, Melinda JM, Crista I, Michael AJ (2002) Abrogation of upstream open reading frame-mediated translational control of a plant S-adenosylmethionine decarboxylase results in polyamine disruption and growth perturbations. J Biol Chem 277:44131-44139 https://doi.org/10.1074/jbc.M206161200
  5. DeScenzo RA, Minocha SC (1993) Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA. Plant Mol Biol 22:113-127 https://doi.org/10.1007/BF00039000
  6. Evans PT, Malmberg RL (1989) Do polyamines have roles in plant development? Annu Rev Plant Mol biol 40:235-269 https://doi.org/10.1146/annurev.pp.40.060189.001315
  7. Franceschetti M, Hanfrey C, Scaramagli S, Torrigiani P, Bagni N, Burtin D, Michael AJ (2001) Characterization of monocot and dicot plant S-adenosyl-L-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames. Biochemical Journal 353:403-409 https://doi.org/10.1042/0264-6021:3530403
  8. Galston AW, Kaur-Sawhney R, Altabella T, Tiburcio AF (1997) Plant polyamines in reproductive activity and response to abiotic stress. Bot Acta 110:197-207 https://doi.org/10.1111/j.1438-8677.1997.tb00629.x
  9. Geballe AP, Morris DR (1994) Initiation codons with 59 leaders of mRNAs as regulators of translation. Trends Biochem Sci 19:159-164 https://doi.org/10.1016/0968-0004(94)90277-1
  10. Geballe AP (1996) Translational control mediated by upstream AUG codons. In: Hershey J, Mathews M, Sonenberg N, (eds), Translational control. Cold spring harbor laboratory press, Cold Spring Harbor, New York, pp 173-197
  11. Hanfrey C, Elliott KA, Franceschetti M, Mayer MJ, Illingworth C, Michael AJ (2005) A dual upstream open reading frame-based autoregulatory circuit controlling polyamine-responsive translation. J Biol Chem 280:39229-39237 https://doi.org/10.1074/jbc.M509340200
  12. Hill JR, Morris DR (1992) Cell-specific translation of S-adenosylmethionine decarboxylase mRNA. J Biol Chem 267:21886-21893
  13. Hinnebusch AG (1990) Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem Sci 15:148-152 https://doi.org/10.1016/0968-0004(90)90215-W
  14. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901-3907
  15. Kakkar RK, Rai VK (1993) Plant polyamines in flowering and fruitripening. Phytochemistry 33:1281-1288 https://doi.org/10.1016/0031-9422(93)85076-4
  16. Kakkar RK, Nagar PK, Ahuja PS, Rai VK (2000) Polyamine and plant morphogenesis. Biol Plant 43:1-11
  17. Kakkar RK, Sawhney VK (2002) Polyamine research in plants-a changing perspective. Physiol Plant 116:281-292 https://doi.org/10.1034/j.1399-3054.2002.1160302.x
  18. Kumar A, Taylor MA, Mad Arif SA, Davies HV (1996) Potato plants expressing antisense and sense S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene: antisense plants display abnormal phenotypes. The Plant J 9:147-158 https://doi.org/10.1046/j.1365-313X.1996.09020147.x
  19. Law GL, Raney A, Heusner C, Morris DR (2001) Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase. J Biol Chem 280:24-28
  20. Lee MM, Lee SH, Park KY (1997) Characterization and expression of two members of the S-adenosylmethionine decarboxylase gene family in carnation flower. Plant Mol Biol 34:371-382 https://doi.org/10.1023/A:1005811229988
  21. Neafsey DE, Galagan JE (2007) Dual modes of natural selection on upstream open reading frames. Mol Biol Evol 24:1744-1751 https://doi.org/10.1093/molbev/msm093
  22. Nyiko T, Sonkoly B, Merai Z, Benkovics AH, Silhavy D (2009) Plant upstream ORFs can trigger nonsense-mediated mRNA decay in a size-dependent manner. Plant Mol Biol 71:367-378 https://doi.org/10.1007/s11103-009-9528-4
  23. Park KY, Lee SH (1994) Effects of ethylene and auxin on polyamine biosynthesis in suspension cultured tobacco cells. Plant Physiol 90:382-390 https://doi.org/10.1111/j.1399-3054.1994.tb00403.x
  24. Raney A, Baron AC, Mize GJ, Law GL, Morris DR (2000) In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase. J Biol Chem 275:24444-24450 https://doi.org/10.1074/jbc.M003364200
  25. Raney A, Law GL, Mize GJ, Morris DR (2002) Regulated translation termination at the upstream open reading frame in S-adenosylmethionine decarboxylase mRNA. J Biol Chem 277:5988-5994 https://doi.org/10.1074/jbc.M108375200
  26. Smith TA (1985) Polyamines. Ann Rev Plant Physiol 36:117-143 https://doi.org/10.1146/annurev.pp.36.060185.001001
  27. Tiburcio AF, Altabella T, Borrell A, Masgrau C (1997) Polyamine metabolism and its regulation. Physiol Plant 100:664-674 https://doi.org/10.1111/j.1399-3054.1997.tb03073.x