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RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis

Nature Plants volume 4, pages 181–188 (2018)Cite this article

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Abstract

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4–siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5′ adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

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Fig. 1: Capturing Pol V-dependent transcripts with GRO–seq.
Fig. 2: Characteristics of Pol V-dependent transcripts.
Fig. 3: Pol V transcripts are sliced in a small-RNA-dependent manner.
Fig. 4: Slicing of Pol V transcripts requires AGO4/6/9.
Fig. 5: Slicing signature of Pol V transcripts is eliminated in spt5l mutants.
Fig. 6: SPT5L is required for slicing of Pol V transcripts.

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Acknowledgements

We thank members of the Jacobsen lab for insightful discussion and M. Akhavan for technical assistance. We also thank Life Science Editors for editing assistance. High-throughput sequencing was performed at UCLA BSCRC BioSequencing Core Facility. W.L. is supported by the Philip J. Whitcome Fellowship from the UCLA Molecular Biology Institute and a scholarship from the Chinese Scholarship Council. Z.Z. is supported by a scholarship from the Chinese Scholarship Council. Group of J.Z. is supported by the Thousand Talents Program for Young Scholars and by the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S172). This work was supported by the NIH grant GM60398 to S.E.J. and NIH grant R01GM094428 and R01GM52413 to J.C. S.E.J. and J.C. are Investigators of the Howard Hughes Medical Institute.

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  1. Wanlu Liu and Sascha H. Duttke contributed equally to this work.

Authors and Affiliations

  1. Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA, USA

    Wanlu Liu & Steven E. Jacobsen

  2. Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA

    Wanlu Liu, Martin Groth, Suhua Feng, Javier Gallego-Bartolome, Zhenhui Zhong, Hsuan Yu Kuo, Jixian Zhai & Steven E. Jacobsen

  3. Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    Sascha H. Duttke, Jonathan Hetzel & Joanne Chory

  4. Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA

    Sascha H. Duttke, Jonathan Hetzel & Joanne Chory

  5. Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA, USA

    Sascha H. Duttke, Jonathan Hetzel & Joanne Chory

  6. Department of Cellular & Molecular Medicine, School of Medicine, University of California at San Diego, La Jolla, CA, USA

    Sascha H. Duttke

  7. Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California at Los Angeles, Los Angeles, CA, USA

    Suhua Feng & Steven E. Jacobsen

  8. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, China

    Zhenhui Zhong & Zonghua Wang

  9. Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen, China

    Jixian Zhai

  10. Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, CA, USA

    Steven E. Jacobsen

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Contributions

W.L., J.H., S.H.D. and S.F. performed the GRO–seq experiments. M.G. performed the ChIP–seq experiments. W.L., J.G.-B., Z.Z. and S.F. performed the small RNA-seq experiments. W.L. and M.G. performed the bioinformatics analysis. W.L. and S.E.J. wrote the manuscript. J.Z., H.Y.K., Z.W. and J.C. assisted in writing the manuscript and discussion.

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Correspondence to Steven E. Jacobsen.

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Supplementary Figures 1–4.

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Supplementary Table 1

Summary of sequenced ChIP-seq, GRO-seq, sRNA-seq and published data used in this paper.

Supplementary Table 2

Genomic location of Pol IV/V-co-dependent sites and Pol IV-independent Pol V sites.

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Liu, W., Duttke, S.H., Hetzel, J. et al. RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis. Nature Plants 4, 181–188 (2018). https://doi.org/10.1038/s41477-017-0100-y

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