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Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning

Nature Genetics volume 38, pages 721–725 (2006)Cite this article

Abstract

Small RNAs have several important biological functions1. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation2. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1–DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

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Figure 1: Generation of Arabidopsis thaliana dcl mutant backgrounds.
Figure 2: Accumulation of miRNAs and trans-acting siRNAs in dcl2 dcl3 dcl4.
Figure 3: siRNA accumulation in dcl mutants.
Figure 4: FWA silencing in dcl mutant backgrounds.
Figure 5: Maintenance of cytosine methylation in dcl mutants.

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Acknowledgements

We thank S. Chan for the dcl2 dcl3 double mutant, J. Carrington for drawing our attention to the IR-71 locus and members of the Jacobsen laboratory for critical reading of the manuscript. I.R.H. was supported by a European Molecular Biology Organization (EMBO) Long Term Postdoctoral Fellowship. S.E.J. is an investigator of the Howard Hughes Medical Institute. This work was supported by US National Science Foundation grants 0439186 and 0548569 to P.J.G. and B.C.M., and NIH grant GM60398 and funds from the Howard Hughes Medical Institute to S.E.J.

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Authors and Affiliations

  1. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 90095, California, USA

    Ian R Henderson, Xiaoyu Zhang, Lianna Johnson & Steven E Jacobsen

  2. Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, 19714, Delaware, USA

    Cheng Lu, Blake C Meyers & Pamela J Green

  3. Howard Hughes Medical Institute, University of California, Los Angeles, 90095, California, USA

    Steven E Jacobsen

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Correspondence to Steven E Jacobsen.

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Supplementary information

Supplementary Fig. 1

Repeat structure and siRNA accumulation at the large inverted repeat IR-71. (PDF 272 kb)

Supplementary Fig. 2

Distribution of methylation at repeat loci. (PDF 200 kb)

Supplementary Table 1

Composition of small RNAs sequenced from wild-type Columbia (Col) and the dcl2-1 dcl3-1 dcl4-2 triple mutant. (PDF 55 kb)

Supplementary Table 2

Sodium bisulfite sequencing analysis of repeat loci. (PDF 44 kb)

Supplementary Table 3

Adaptor, primer and probe sequences. (PDF 43 kb)

Supplementary Table 4

Small RNA sequences cloned from Columbia that show a perfect match to the Arabidopsis genome. (PDF 417 kb)

Supplementary Table 5

Small RNA sequences cloned from dcl2 dcl3 dcl4 that show a perfect match to the Arabidopsis genome. (PDF 285 kb)

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Henderson, I., Zhang, X., Lu, C. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38, 721–725 (2006). https://doi.org/10.1038/ng1804

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