Skip to main content
SpringerLink
Log in

Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.) cells. Among the different phases of the cell cycle, transgenic cells at the M phase gave 2–3 times lower gfp silencing than those at the G1, S, and G2 phases. The similar results of the siRNA-mediated gfp silencing were obtained in three transgenic cell lines. Differential gfp silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that siRNA-mediated gene inactivation is associated with the cell cycle phase in Virginia pine.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

GFP:

Green fluorescent protein

35S:

Cauliflower mosaic virus 35S promoter

siRNA:

Short interfering RNA

References

  • Akashi H, Miyagishi M, Taira K (2001) Suppression of gene expression by RNA interference in cultured plant cells. Antisense Nucleic Acid Drug Dev 11:359–367

    Article  PubMed  CAS  Google Scholar 

  • Amedeo P, Habu Y, Afsar K, Scheid OM, Paszkowski J (2000) Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 405:203–206

    Article  PubMed  CAS  Google Scholar 

  • Bartee L, Malagnac F, Bender J (2001) Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev 15:1753–1758

    Article  PubMed  CAS  Google Scholar 

  • Bettinger T, Carlisle RC, Read ML, Ogris M, Seymour LW (2001) Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells. Nucleic Acids Res 29:3882–3891

    Article  PubMed  CAS  Google Scholar 

  • Callebaut I, Courvalin JC, Mornon JP (1999) The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation. FEBS Lett 446:189–193

    Article  PubMed  CAS  Google Scholar 

  • Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990

    Article  PubMed  CAS  Google Scholar 

  • Cogoni C, Macino G (2000) Post-transcriptional gene silencing across kingdoms. Curr Opin Genet Dev 10:638–643

    Article  PubMed  CAS  Google Scholar 

  • Curradi M, Izzo A, Badaracco G, Landsberger N (2002) Molecular mechanisms of gene silencing mediated by DNA methylation. Mol Cell Biol 22:3157–3173

    Article  PubMed  CAS  Google Scholar 

  • Elbashir SM, Ledeckel W, Yalcin A, Weber K, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200

    Article  PubMed  CAS  Google Scholar 

  • Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  PubMed  CAS  Google Scholar 

  • Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci USA 101:1927–1932

    Article  PubMed  CAS  Google Scholar 

  • Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    Article  PubMed  CAS  Google Scholar 

  • Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296

    Article  PubMed  CAS  Google Scholar 

  • Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127

    Article  PubMed  CAS  Google Scholar 

  • Holen T, Amarzguioui M, Wiiger MT, Babaie E, Prydz H (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res 30:1757–1766

    Article  PubMed  CAS  Google Scholar 

  • Hutvagner G, Zarmore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12:225–232

    Article  PubMed  CAS  Google Scholar 

  • Iida A, Yamashita T, Yamada Y, Morikawa H (1991) Efficiency of particle-bombardment-mediated transformation is influenced by cell-cycle stage in synchronized cultured-cells of tobacco. Plant Physiol 97:1585–1587

    PubMed  CAS  Google Scholar 

  • Kawata T, Nakayama T, Ohtsubo N, Tabata T, Iwabuchi M (1990) Cell cycle-regulated gene expression in transgenic plant cells. Dev Genet 11:205–213

    Article  PubMed  CAS  Google Scholar 

  • Klahre U, Crete P, Leuenberger SA, Iglesias VA, Meins F (2002) High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Proc Natl Acad Sci USA 99:11981–11986

    Article  PubMed  CAS  Google Scholar 

  • Lipardi C, Wei Q, Paterson BM (2001) RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107:297–307

    Article  PubMed  CAS  Google Scholar 

  • Misquitta L, Paterson BM (1999) Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA 96:1451–1456

    Article  PubMed  CAS  Google Scholar 

  • Montgomery MK, Xu S, Fire A (1998) RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 95:15502–15507

    Article  PubMed  CAS  Google Scholar 

  • Nagata T, Okada K, Kawazu T, Takebe I (1987) Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells. Mol Genet Genomics 207:242–244

    Article  CAS  Google Scholar 

  • Ngo H, Tschudi C, Gull K, Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 95:14687–14692

    Article  PubMed  CAS  Google Scholar 

  • Nishikura K (2001) A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107:415–418

    Article  PubMed  CAS  Google Scholar 

  • Nykanen A, Haley B, Zarmore PD (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci USA 101:1892–1897

    Article  PubMed  CAS  Google Scholar 

  • Schwarze SR, Ho A, Vocero-Akbani A, Dowdy S (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569–1572

    Article  PubMed  CAS  Google Scholar 

  • Scott A, Wyatt S, Tsou P-L, Robertson D, Strömgren-Allen N (1999) Model system for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26:1125–1132

    PubMed  CAS  Google Scholar 

  • Semizarov D, Frost L, Sarthy A, Kroeger P, Halbert DN, Fesik SW (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA 100:6347–6352

    Article  PubMed  CAS  Google Scholar 

  • Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

    Article  PubMed  CAS  Google Scholar 

  • Stewart CN (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382

    Article  PubMed  CAS  Google Scholar 

  • Tabara H, Grishok A, Mello CC (1998) RNAi in C. elegans: soaking in the genome sequence. Science 282:430–431

    Article  PubMed  CAS  Google Scholar 

  • Tang W, Tian YC (2003) Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud. J Exp Bot 54:835–844

    Article  PubMed  CAS  Google Scholar 

  • Tang W, Sederoff R, Whetten R (2001) Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens. Planta 21(3):981–989

    Google Scholar 

  • Van Houdt H, Bleys A, Depicker A (2003) RNA target sequences promote spreading of RNA silencing. Plant Physiol 131:245–253

    Article  PubMed  CAS  Google Scholar 

  • Vanithatani R, Chellappan P, Fauquet CM (2003) Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci USA 100:9632–9636

    Article  PubMed  CAS  Google Scholar 

  • Volker A, Stierhof Y-D, Jurgens G (2001) Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis. J Cell Sci 114:3001–3012

    PubMed  CAS  Google Scholar 

  • Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature 411:834–842

    Article  PubMed  CAS  Google Scholar 

  • Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson S, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590

    Article  PubMed  CAS  Google Scholar 

  • Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2:70–75

    Article  PubMed  CAS  Google Scholar 

  • Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. C.N. Stewart and Dr. J. Haseloff for providing us with the m-gfp5-ER constructs, Dr. R. Qu for the facilities used for particle bombardment for part of the work, and A. Smith (The Flow Cytometry-Confocal Microscopy Core Facility, East Carolina University, USA) for technical assistance with confocal microscopy for quantitative analysis of green fluorescence.

Author information

Authors and Affiliations

  1. Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC, 27858-4353, USA

    Wei Tang & Ronald J. Newton

  2. Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC, 27858-4354, USA

    Douglas A. Weidner

Authors
  1. Wei Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ronald J. Newton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas A. Weidner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Tang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tang, W., Newton, R.J. & Weidner, D.A. Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase. Planta 224, 53–60 (2006). https://doi.org/10.1007/s00425-005-0190-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-005-0190-z

Keywords

Search

Navigation