Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation.
Vyšlo v časopise: A Pre-mRNA-Splicing Factor Is Required for RNA-Directed DNA Methylation in. PLoS Genet 9(9): e32767. doi:10.1371/journal.pgen.1003779 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003779
1. LischD, BennetzenJL (2011) Transposable element origins of epigenetic gene regulation. Current Opinion in Plant Biology 14 : 156–161.
2. MirouzeM, PaszkowskiJ (2011) Epigenetic contribution to stress adaptation in plants. Current Opinion in Plant Biology 14 : 267–274.
3. HeardE (2004) Recent advances in X-chromosome inactivation. Current Opinion in Cell Biology 16 : 247–255.
4. ErhardKF, HollickJB (2011) Paramutation: a process for acquiring trans-generational regulatory states. Current Opinion in Plant Biology 14 : 210–216.
5. BauerMJ, FischerRL (2011) Genome demethylation and imprinting in the endosperm. Current Opinion in Plant Biology 14 : 162–167.
6. CokusSJ, FengSH, ZhangXY, ChenZG, MerrimanB, et al. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452 : 215–219.
7. SazeH, ScheidOM, PaszkowskiJ (2003) Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nature Genetics 34 : 65–69.
8. HenikoffS, ComaiL (1998) A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149 : 307–318.
9. CaoXF, JacobsenSE (2002) Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proceedings of the National Academy of Sciences of the United States of America 99 : 16491–16498.
10. ChanSWL, HendersonIR, JacobsenSE (2005) Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Reviews Genetics 6 : 351–360.
11. WasseneggerM, HeimesS, RiedelL, SangerHL (1994) Rna-Directed De-Novo Methylation of Genomic Sequences in Plants. Cell 76 : 567–576.
12. MatzkeM, KannoT, ClaxingerL, HuettelB, MatzkeAJM (2009) RNA-mediated chromatin-based silencing in plants. Current Opinion in Cell Biology 21 : 367–376.
13. VaucheretH (2005) RNA polymerase IV and transcriptional silencing. Nature Genetics 37 : 659–660.
14. HerrAJ, JensenMB, DalmayT, BaulcombeDC (2005) RNA polymerase IV directs silencing of endogenous DNA. Science 308 : 118–120.
15. KasschauKD, FahlgrenN, ChapmanEJ, SullivanCM, CumbieJS, et al. (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. Plos Biology 5 : 479–493.
16. XieZX, JohansenLK, GustafsonAM, KasschauKD, LellisAD, et al. (2004) Genetic and functional diversification of small RNA pathways in plants. Plos Biology 2 : 642–652.
17. LiJJ, YangZY, YuB, LiuJ, ChenXM (2005) Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabildopsis. Current Biology 15 : 1501–1507.
18. YuB, YangZY, LiJJ, MinakhinaS, YangMC, et al. (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307 : 932–935.
19. SmithLM, PontesO, SearleL, YelinaN, YousafzaiFK, et al. (2007) An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19 : 1507–1521.
20. LiuJ, BaiG, ZhangCJ, ChenW, ZhouJX, et al. (2011) An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing. Cell Research 21 : 1691–1700.
21. LawJA, VashishtAA, WohlschlegelJA, JacobsenSE (2011) SHH1, a Homeodomain Protein Required for DNA Methylation, As Well As RDR2, RDM4, and Chromatin Remodeling Factors, Associate with RNA Polymerase IV. Plos Genetics 7: e1002195.
22. QiYJ, HeXY, WangXJ, KohanyO, JurkaJ, et al. (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443 : 1008–1012.
23. WierzbickiAT, ReamTS, HaagJR, PikaardCS (2009) RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nature Genetics 41 : 630–634.
24. LawJA, AusinI, JohnsonLM, VashishtAA, ZhuJK, et al. (2010) A Protein Complex Required for Polymerase V Transcripts and RNA-Directed DNA Methylation in Arabidopsis. Current Biology 20 : 951–956.
25. HaagJR, PikaardCS (2011) Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nature Reviews Molecular Cell Biology 12 : 483–492.
26. LiCF, PontesO, El-ShamiM, HendersonIR, BernatavichuteYV, et al. (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126 : 93–106.
27. El-ShamiM, PontierD, LahmyS, BraunL, PicartC, et al. (2007) Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes & Development 21 : 2539–2544.
28. HeXJ, HsuYF, ZhuSH, WierzbickiAT, PontesO, et al. (2009) An Effector of RNA-Directed DNA Methylation in Arabidopsis Is an ARGONAUTE 4-and RNA-Binding Protein. Cell 137 : 498–508.
29. GaoZH, LiuHL, DaxingerL, PontesO, HeXJ, et al. (2010) An RNA polymerase II - and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465 : 106–U118.
30. ZhengBL, WangZM, LiSB, YuB, LiuJY, et al. (2009) Intergenic transcription by RNA Polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes & Development 23 : 2850–2860.
31. HaveckerER, WallbridgeLM, HardcastleTJ, BushMS, KellyKA, et al. (2010) The Arabidopsis RNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci. Plant Cell 22 : 321–334.
32. ZhengXW, ZhuJH, KapoorA, ZhuJK (2007) Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. Embo Journal 26 : 1691–1701.
33. HendersonIR, DelerisA, WongW, ZhongXH, ChinHG, et al. (2010) The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA-Directed DNA Methylation in Arabidopsis thaliana. Plos Genetics 6: e1001182.
34. LorkovicZJ, NaumannU, MatzkeAJM, MatzkeM (2012) Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana. Current Biology 22 : 933–938.
35. MoissiardG, CokusSJ, CaryJ, FengS, BilliAC, et al. (2012) MORC family ATPases required for heterochromatin condensation and gene silencing. Science 336 : 1448–1451.
36. AusinI, GreenbergMVC, LiCF, JacobsenSE (2012) The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis. Epigenetics 7 : 29–33.
37. JuricaMS, MooreMJ (2003) Pre-mRNA splicing: Awash in a sea of proteins. Molecular Cell 12 : 5–14.
38. KramerA (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annual Review of Biochemistry 65 : 367–409.
39. GolovkinM, ReddyASN (1999) An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein. Journal of Biological Chemistry 274 : 36428–36438.
40. TanabeN, KimuraA, YoshimuraK, ShigeokaS (2009) Plant-specific SR-related protein atSR45a interacts with spliceosomal proteins in plant nucleus. Plant Molecular Biology 70 : 241–252.
41. GongZH, Morales-RuizT, ArizaRR, Roldan-ArjonaT, DavidL, et al. (2002) ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111 : 803–814.
42. HeXJ, HsuYF, ZhuSH, LiuHL, PontesO, et al. (2009) A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development. Genes & Development 23 : 2717–2722.
43. HeXJ, HsuYF, PontesO, ZhuJH, LuJ, et al. (2009) NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes & Development 23 : 318–330.
44. ZhengZM, XingY, HeXJ, LiWB, HuYL, et al. (2010) An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis. Plant Journal 62 : 92–99.
45. ZhuJ, KapoorA, SridharVV, AgiusF, ZhuJK (2007) The DNA glycosylase/lyase ROS1 functions in pruning DNA methylation patterns in Arabidopsis. Current Biology 17 : 54–59.
46. ChakarovaCF, HimsMM, BolzH, Abu-SafiehL, PatelRJ, et al. (2002) Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa. Human Molecular Genetics 11 : 87–92.
47. AnthonyJG, WeidenhammerEM, WoolfordJL (1997) The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP. Rna-a Publication of the Rna Society 3 : 1143–1152.
48. WierzbickiAT, HaagJR, PikaardCS (2008) Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135 : 635–648.
49. ZhangCJ, ZhouJX, LiuJ, MaZY, ZhangSW, et al. (2013) The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis. Embo Journal 32 : 1128–1140.
50. LeeBH, KapoorA, ZhuJ, ZhuJK (2006) STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis. Plant Cell 18 : 1736–1749.
51. MoazedD (2009) Small RNAs in transcriptional gene silencing and genome defence. Nature 457 : 413–420.
52. GrewalSI (2010) RNAi-dependent formation of heterochromatin and its diverse functions. Current Opinion in Genetics & Development 20 : 134–141.
53. BayneEH, PortosoM, KaganskyA, Kos-BraunIC, UranoT, et al. (2008) Splicing factors facilitate RNAi-directed silencing in fission yeast. Science 322 : 602–606.
54. ChinenM, MoritaM, FukumuraK, TaniT (2010) Involvement of the spliceosomal U4 small nuclear RNA in heterochromatic gene silencing at fission yeast centromeres. J Biol Chem 285 : 5630–5638.
55. LiuYG, MitsukawaN, OosumiT, WhittierRF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8 : 457–463.
56. AudicS, ClaverieJM (1997) The significance of digital gene expression profiles. Genome Research 7 : 986–995.
57. HarrisEY, PontsN, Le RochKG, LonardiS (2012) BRAT-BW: efficient and accurate mapping of bisulfite-treated reads. Bioinformatics 28 : 1795–1796.
58. QianW, MikiD, ZhangH, LiuY, ZhangX, et al. (2012) A histone acetyltransferase regulates active DNA demethylation in Arabidopsis. Science 336 : 1445–1448.
59. SalehA, Alvarez-VenegasR, AvramovaZ (2008) An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3 : 1018–1025.
60. YooSD, ChoYH, SheenJ (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2 : 1565–1572.
61. OnoderaY, HaagJR, ReamT, NunesPC, PontesO, et al. (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120 : 613–622.
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