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

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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.

References

  1. 1.

    Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

  2. 2.

    Blevins, T. et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. eLife 4, e09591 (2015).

  3. 3.

    Zhai, J. et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163, 445–455 (2015).

  4. 4.

    Li, S. et al. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis. Genome Res. 25, 235–245 (2015).

  5. 5.

    Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS. Biol. 2, E104 (2004).

  6. 6.

    Haag, J. R. et al. In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol. Cell. 48, 811–818 (2012).

  7. 7.

    Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell. 19, 421–428 (2005).

  8. 8.

    Zilberman, D., Cao, X. & Jacobsen, S. E. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716–719 (2003).

  9. 9.

    Li, C. F. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006).

  10. 10.

    Qi, Y. et al. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012 (2006).

  11. 11.

    Wierzbicki, A. T., Haag, J. R. & Pikaard, C. S. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135, 635–648 (2008).

  12. 12.

    Zhong, X. et al. Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157, 1050–1060 (2014).

  13. 13.

    Böhmdorfer, G. et al. Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin. eLife 5, e19092 (2016).

  14. 14.

    Wierzbicki, A. T., Ream, T. S., Haag, J. R. & Pikaard, C. S. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41, 630–634 (2009).

  15. 15.

    Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

  16. 16.

    Hetzel, J., Duttke, S. H., Benner, C. & Chory, J. Nascent RNA sequencing reveals distinct features in plant transcription. Proc. Natl Acad. Sci. USA 113, 12316–12321 (2016).

  17. 17.

    Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).

  18. 18.

    Zhong, X. et al. DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons. Nat. Struct. Mol. Biol. 19, 870–875 (2012).

  19. 19.

    Johnson, L. M. et al. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507, 124–128 (2014).

  20. 20.

    Smale, S. T. & Kadonaga, J. T. The RNA polymerase II core promoter. Annu. Rev. Biochem. 72, 449–479 (2003).

  21. 21.

    Sollner-Webb, B. & Reeder, R. H. The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell 18, 485–499 (1979).

  22. 22.

    Zecherle, G. N., Whelen, S. & Hall, B. D. Purines are required at the 5′ ends of newly initiated RNAs for optimal RNA polymerase III gene expression. Mol. Cell. Biol. 16, 5801–5810 (1996).

  23. 23.

    El-Shami, M. et al. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes. Dev. 21, 2539–2544 (2007).

  24. 24.

    Mi, S. et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008).

  25. 25.

    Havecker, E. R. et al. The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22, 321–334 (2010).

  26. 26.

    Wang, H. et al. Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions. Plant J. 67, 292–304 (2011).

  27. 27.

    Vo Ngoc, L., Cassidy, C. J., Huang, C. Y., Duttke, S. H. C. & Kadonaga, J. T. The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters. Genes. Dev. 31, 6–11 (2017).

  28. 28.

    Eun, C. et al. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana. PLoS ONE 6, e25730 (2011).

  29. 29.

    Wang, F. & Axtell, M. J. AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V-dependent loci in Arabidopsis thaliana. Plant J. 90, 37–47 (2017).

  30. 30.

    He, X.-J. et al. An effector of RNA-directed DNA methylation in Arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137, 498–508 (2009).

  31. 31.

    Rowley, M. J., Avrutsky, M. I., Sifuentes, C. J., Pereira, L. & Wierzbicki, A. T. Independent chromatin binding of ARGONAUTE4 and SPT5L/KTF1 mediates transcriptional gene silencing. PLoS Genet 7, e1002120 (2011).

  32. 32.

    Bies-Etheve, N. et al. RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family. EMBO Rep. 10, 649–654 (2009).

  33. 33.

    Greenberg, M. V. C. et al. Identification of genes required for de novo DNA methylation in Arabidopsis. Epigenetics 6, 344–354 (2011).

  34. 34.

    Huang, L. et al. An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II. Nat. Struct. Mol. Biol. 16, 91–93 (2009).

  35. 35.

    Zhong, X. et al. Domains rearranged methyltransferase3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis. Proc. Natl Acad. Sci. USA 112, 911–916 (2015).

  36. 36.

    Ausin, I., Mockler, T. C., Chory, J. & Jacobsen, S. E. IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana. Nat. Struct. Mol. Biol. 16, 1325–1327 (2009).

  37. 37.

    Ausin, I. et al. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis.Proc. Natl Acad. Sci. USA 109, 8374–8381 (2012).

  38. 38.

    Zhang, C.-J. et al. IDN2 and its paralogs form a complex required for RNA-directed DNA methylation. PLoS. Genet. 8, e1002693 (2012).

  39. 39.

    Groth, M. et al. SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation. Proc. Natl Acad. Sci. USA 111, 17666–17671 (2014).

  40. 40.

    Stroud, H., Greenberg, M. V. C., Feng, S., Bernatavichute, Y. V. & Jacobsen, S. E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).

  41. 41.

    Han, Y.-F. et al. SUVR2 is involved in transcriptional gene silencing by associating with SNF2-related chromatin-remodeling proteins in Arabidopsis. Cell. Res. 24, 1445–1465 (2014).

  42. 42.

    Lahmy, S. et al. Evidence for ARGONAUTE4–DNA interactions in RNA-directed DNA methylation in plants. Genes. Dev. 30, 2565–2570 (2016).

  43. 43.

    Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007).

  44. 44.

    Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007).

  45. 45.

    Shimada, Y., Mohn, F. & Bühler, M. The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts. Genes. Dev. 30, 2571–2580 (2016).

  46. 46.

    Noma, K.-I. et al. RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat. Genet. 36, 1174–1180 (2004).

  47. 47.

    Zofall, M. et al. RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335, 96–100 (2012).

  48. 48.

    Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).

  49. 49.

    Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes. Dev. 19, 2030–2040 (2005).

  50. 50.

    Ream, T. S. et al. Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol. Cell. 33, 192–203 (2009).

  51. 51.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  52. 52.

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

  53. 53.

    Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genom. 15, 284 (2014).

  54. 54.

    Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

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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.

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 Information

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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