Volume 54, Issue 3, Pages 418-430 (May 2014) An Rrp6-like Protein Positively Regulates Noncoding RNA Levels and DNA Methylation in Arabidopsis  Huiming Zhang, Kai Tang, Weiqiang Qian, Cheng-Guo Duan, Bangshing Wang, Heng Zhang, Pengcheng Wang, Xiaohong Zhu, Zhaobo Lang, Yu Yang, Jian-Kang Zhu  Molecular Cell  Volume 54, Issue 3, Pages 418-430 (May 2014) DOI: 10.1016/j.molcel.2014.03.019 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 AtRRP6L1 Is an Epigenetic Regulator (A) Chop-PCR detection of DNA methylation levels at the AtSN1 locus. The methylation-sensitive restriction enzyme was HaeIII. Nondigested controls are shown below. (B) RT-qPCR measurements of AtSN1 RNA levels. (C) Chop-PCR examination of DNA methylation at a group of TE loci. Restriction enzymes HaeIII, HpyCH4 IV, BstB I, and BsmF I are indicted on the right. The examined loci are indicated on the left. (D) Bisulfite sequencing quantification of DNA methylation levels at At1TE40810 and At5TE27040 loci. Cytosines were examined as CG, CHG, CHH, and total that indicates the sum of all three types of cytosines (x axis). The methylation levels (y axis) indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined regions. The gray-scaled pie charts indicate the proportions of CG, CHG, and CHH within the examined regions that cover the two short TEs. (E) Chromatin immunoprecipitation (ChIP) measurements of histone H3K9me2 levels. (F) RT-qPCR quantification of mRNA levels of genes neighboring AtRRP6L1-regulated TEs. Error bars indicate SD, n ≥ 3. See also Figure S1. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 AtRRP6L1 Controls Genome-wide RdDM Independently of Exosomal RNA Degradation (A) Heatmap depiction of the 2,261 loci where AtRRP6L1 dysfunction causes DNA hypomethylation. Each hypomethylated region corresponds to a colored horizontal bar and the bars are clustered numerically into a column (y axis). Cytosines were examined as CG, CHG, and CHH. The color-scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions. (B) The proportions of three different C (CG, CHG, or CHH) contexts to all hypomethylated C (CG + CHG + CHH) in the mutants. The ratios of each C context (y axis) are presented in a stacked bar that adds up to 100% of all the hypomethylated C in each mutant (x axis). (C) Overlapping patterns among the genome-wide hypomethylation loci in atrrp6l1-2, nrpd1-3, and nrpe1-11. (D) AtRRP6L2 mutations do not phenocopy DNA hypomethylation observed in atrrp6l1 mutants. Chop-PCR results are shown. Restriction enzymes HaeIII, HpyCH4 IV, and BstB I are indicated on the left. The examined loci are indicated on the right. (E) Knockdown of RRP41 in the rrp41iRNAi by estradiol (Est) treatments causes severe growth retardation. (F) The exosome substrate pre-miR156e accumulates in Est-treated rrp41iRNAi. RT-qPCR results are shown. Error bars indicate SD, n = 3. (G) DNA methylation levels at AtRRP6L1 target loci in Est-treated rrp41iRNAi. Chop-PCR results are shown. Restriction enzymes HaeIII, HpyCH4 IV, and BstB I are indicted on the left. The examined loci are indicated on the right. See also Figure S2. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 AtRRP6L1 Mutations Impair Accumulation of Pol V Transcripts at Target Loci (A) Quantification of AtSN1 scaffold RNAs by gene-specific RT-qPCR. (B) Examination of possible aberrant transcriptional readthrough from AtSN1 upstream regions. Positions of the qPCR amplicons are as indicated. (C) DNA methylation and NRPE1 occupancy at UP556840. DNA methylation levels and NRPE1 occupancy are snapshotted from whole-genome bisulfite sequencing results and from Wierzbicki et al. (2012), respectively. (D) RT-qPCR measurements of UP556840 RNAs. (E) DNA methylation and NRPE1 occupancy at DN400413. (F) RT-qPCR measurements of DN400413 RNAs. Error bars indicate SD, n ≥ 3. See also Figure S3. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 AtRRP6L1 Associates with Scaffold RNAs and Partially Colocalizes with Pol V in Subnuclear Foci (A) AtRRP6L1 binds to RNA in vitro as shown by EMSA. The N-terminal half (N) and C-terminal half (C) of GST-fused AtRRP6L1 proteins were used. (B) AtRRP6L1 associates with scaffold RNAs in vivo as shown by RNA IP using atrrp6l1-2/AtRRP6L1-HA and Col-0 plants. Transcripts were detected by gene-specific RT-PCR after DNase treatment. Total RNA controls, assayed from input samples without IP, show that the RNAs are present in equivalent amounts in Col-0 and atrrp6l1-2/AtRRP6L1-HA plants. No RT controls used SN1C primers. Background signals of Actin2 RNA show that equal RNA amounts from the IP fractions were tested. (C) AtRR6L1 colocalizes with NRPE1 in the perinucleolar dot in 38% of the nuclei that show a colocalization or partial colocalization of the two proteins (upper panel). Partial colocalization was also observed in discrete nucleoplasmic foci in 62% of the nuclei that show a colocalization or partial colocalization of the two proteins (lower panel). The majority (55%) of 158 nuclei examined in four biological replicates showed a colocalization or partial colocalization of the two proteins. NRPE1 (red) is localized by its specific antibody in cells expressing HA-tagged AtRRP6L1 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein colocalization. DNA (blue) was stained with DAPI. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 AtRRP6L1 Promotes the Retention of Pol V Transcripts in Chromatin (A) Schematic procedure of nuclei fractionation. Nuclei are lysed in the presence of urea and detergent, followed by centrifugation that sediments chromatin-associated RNAs in the pellet (chromatin-associated fraction), while leaving released RNAs in the supernant (chromatin-free fraction). Genomic DNAs are nearly absent in the chromatin-free fractions, as examined by qPCR (Figure S3E). (B) Gene-specific RT-qPCR measurements of Pol V-transcribed noncoding RNAs and a housekeeping gene, SKP1, in the chromatin-associated fractions. RNA levels are relative to Actin2 in the same RNA samples. No RT controls using UP566840 primers detected no PCR signals. (C) Gene-specific RT-qPCR measurements of Pol V-transcribed noncoding RNAs and a housekeeping gene, SKP1, in the chromatin-free fractions. (D) Detection of Pol V occupancy in chromatin by ChIP assays using anti-NRPE1 antibody. Error bars indicate SD, n = 3. See also Figure S3. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 AtRRP6L1 Regulates Pol IV-Dependent Small RNA Production (A) Quantification of individual 24 nt siRNAs by TaqMan small RNA assays. Sequences of the siRNAs are listed in Table S3. Error bars indicate SD, n ≥ 3. (B) AtRRP6L1-dependent small RNAs are dominated by 23 nt and 24 nt siRNAs. Loci with small RNAs of very low abundance (≤10 HNA, see Supplemental Information for details) were indicated by white color. (C) The AtRRP6L1-regulated siRNA loci show dependence on Pol IV and, to a lesser degree, on Pol V. (D) AtRRP6L1 does not control siRNAs at the IR71, TAS1, and TAS2 loci. Vertical gray bars indicate the siRNA sequencing coverage normalized to the same scale in WT and the mutant. See also Figures S4 and S5. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 A Model of AtRRP6L1 Function in RNA-Directed DNA Methylation At RdDM target loci where DNA methylation is AtRRP6L1 dependent, AtRRP6L1 retains Pol V-generated noncoding RNAs at sites of transcription, allowing for prolonged association of transcripts with chromatin for their scaffold function. Since AtRRP6L1 is also required for Pol IV-dependent siRNA production, AtRRP6L1 possibly also helps to retain Pol IV-transcribed noncoding RNAs, thereby facilitating synthesis of double-stranded RNAs by RDR2 and subsequent production of 24 nt siRNAs. In the absence of AtRRP6L1, these important noncoding RNAs in the RdDM pathway may be quickly released from the chromatin upon transcription termination, resulting in DNA hypomethylation. For simplicity, not all known RdDM components are shown. Molecular Cell 2014 54, 418-430DOI: (10.1016/j.molcel.2014.03.019) Copyright © 2014 Elsevier Inc. Terms and Conditions