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

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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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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.

Author information

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.

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) doi:10.1038/s41477-017-0100-y

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