Skip to main content
SpringerLink
Log in

The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3 trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis.

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

Similar content being viewed by others

Abbreviations

ARF:

Auxin response factor

ATA:

Aurintricarboxylic acid

DCL:

Dicer-like protein

DRB:

dsRNA-binding protein

dsRBM:

dsRNA-binding motif

dsRNA:

Double-stranded RNA

GFP:

Green fluorescent protein

GST:

Glutathion S-transferase

HA:

Hemagglutinin

HYL1:

HYPONASTIC LEAVES1

LNA:

Locked nucleic acid

miRNA:

microRNAs

RNAi:

RNA interference

siRNA:

small interfering RNA

ta-siRNA:

trans-acting siRNA

TRBP:

TAR RNA-binding protein

WT:

Wild-type

References

  • Adenot X, Elmayan T, Lauressergues D, Boutet S, Bouche N, Gasciolli V, Vaucheret H (2006) DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr Biol 16:927–932

    Article  PubMed  CAS  Google Scholar 

  • Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2006) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–1291

    Article  CAS  Google Scholar 

  • Bouche N, Lauressergues D, Gasciolli V, Vaucheret H (2006) An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J 25:3347–3356

    Article  PubMed  CAS  Google Scholar 

  • Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhattar R (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436:740–744

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313:68–71

    Article  PubMed  CAS  Google Scholar 

  • Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235

    Article  PubMed  CAS  Google Scholar 

  • Forstemann K, Tomari Y, Du T, Vagin VV, Denli AM, Bratu DP, Klattenhoff C, Theurkauf WE, Zamore PD (2005) Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 3:E236

    Article  PubMed  CAS  Google Scholar 

  • Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA, Watson JM, Wang MB, Brosnan C, Carroll BJ, Waterhouse PM (2006) RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 7:1168–1175

    Article  PubMed  CAS  Google Scholar 

  • Gasciolli V, Mallory AC, Bartel DP, Vaucheret H (2005) Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol 15:1494–1500

    Article  PubMed  CAS  Google Scholar 

  • Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432:235–240

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Han MH, Goud S, Song L, Fedoroff N (2004) The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci USA 101:1093–1098

    Article  PubMed  CAS  Google Scholar 

  • Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, Jacobsen SE (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38:721–725

    Article  PubMed  CAS  Google Scholar 

  • Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T (2005) Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57:173–188

    Article  PubMed  CAS  Google Scholar 

  • Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101:12753–12758

    Article  PubMed  CAS  Google Scholar 

  • Kurihara Y, Takashi Y, Watanabe Y (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12:206–212

    Article  PubMed  CAS  Google Scholar 

  • Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X (2003) R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301:1921–1925

    Article  PubMed  CAS  Google Scholar 

  • Mancinelli AL (1990) Interaction between light quality and light quantity in the photoregulation of anthocyanin production. Plant Physiol 92:1191–1195

    Article  PubMed  CAS  Google Scholar 

  • Miyazaki S, Koga R, Bohnert HJ, Fukuhara T (1999) Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum. Mol Gen Genet 261:307–316

    Article  PubMed  CAS  Google Scholar 

  • Nagy F, Kay SA, Chua NH (1988) Analysis of gene expression in transgenic plants. Plant Mol Biol Manual B4:1–29

    Google Scholar 

  • Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12:1484–1495

    Article  PubMed  CAS  Google Scholar 

  • Saito K, Ishizuka A, Siomi H, Siomi MC (2005) Processing of pre-microRNAs by the dicer-1-loquacious complex in Drosophila cells. PLoS Biol 3:E235

    Article  PubMed  CAS  Google Scholar 

  • Schauer SE, Jacobsen SE, Meinke DW, Ray A (2002) DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci 7:487–491

    Article  PubMed  CAS  Google Scholar 

  • Tabara H, Yigit E, Siomi H, Mello CC (2002) The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109:861–871

    Article  PubMed  CAS  Google Scholar 

  • Vazquez F, Gasciolli V, Crete P, Vaucheret H (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr Biol 14:346–351

    Article  PubMed  CAS  Google Scholar 

  • Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, Havelda Z (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32(22):e175

    Article  PubMed  CAS  Google Scholar 

  • Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104

    Article  PubMed  Google Scholar 

  • Xie Z, Allen E, Wilken A, Carrington JC (2005) DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc Natl Acad Sci USA 102:12984–12989

    Article  PubMed  CAS  Google Scholar 

  • Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Genomanalyse im Biologischen System Pflanze and the Arabidopsis Biological Resource Center for kindly providing the Arabidopsis seeds, and Dr. Masahiro Kasahara, Tokyo University of Agriculture and Technology, Tokyo, Japan, for kindly providing the plasmids. This research was supported in part by Grants-in-Aid for Scientific Research (No. 17657078 to T.F. and No.18–1297 to A.H.) from the Ministry of Education, Science, Sports, Culture and Technology of Japan, and a grant for protein network research from the Nara Institute of Science and Technology to A.H.

Author information

Authors and Affiliations

  1. Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo, 183-8509, Japan

    Yukihiro Nakazawa, Akihiro Hiraguri, Hiromitsu Moriyama & Toshiyuki Fukuhara

Authors
  1. Yukihiro Nakazawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akihiro Hiraguri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiromitsu Moriyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Toshiyuki Fukuhara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiyuki Fukuhara.

Additional information

Yukihiro Nakazawa and Akihiro Hiraguri contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material

ESM1 (DOC 58 kb)

ESM2 (PPT 4,013 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nakazawa, Y., Hiraguri, A., Moriyama, H. et al. The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway. Plant Mol Biol 63, 777–785 (2007). https://doi.org/10.1007/s11103-006-9125-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-006-9125-8

Keywords

Search

Navigation