A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis

Abstract

DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. 24-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ∼ 20-60 nt in length, often having the same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis. Precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

Figure 1. Identification of sidRNAs in Arabidopsis

(A) Pie chart summarizing the numbers of sidRNA loci in the indicated categories. (B) Heatmap showing the size distribution of sequenced sRNAs from sidRNA-producing loci in Col-0, dcl2/3/4 and dcl1/2/3/4. Color intensity represents the fraction of sRNAs of different sizes. (C) Scatter plot showing the abundance (log2RPM) of each miRNA (red) and sidRNA locus (blue) in dcl1/2/3/4 versus that in dcl2/3/4. A reference line of slope=1 is shown. The Spearman’s correlation coefficient (ρ) for sidRNAs in dcl1/2/3/4 and dcl2/3/4 mutants is indicated. (D) Box plots of levels of sRNAs of the indicated sizes produced from sidRNA loci in Col-0, dcl2/3/4 and dcl1/2/3/4. Asterisks indicate a significant difference between Col-0 and the mutants (P < 10⁻¹⁵, Mann–Whitney U test). (E) Box plots of the percentages of laddered sRNAs produced from sidRNA loci in Col-0, dcl2/3/4 and dcl1/2/3/4. Asterisks indicate a significant difference between Col-0 and the mutants (P < 10⁻¹⁵, Mann–Whitney U test). (F) A representative group of laddered sRNAs produced from AtREP2. Numbers represent the abundance (RPM) of sRNAs with different lengths ranging from 19 to 29 nt. Values in the parentheses indicate the percentages of sRNAs with different lengths. (G) Detection of sRNA production at representative sidRNA loci in the indicated plants by Northern blot. 5S rRNAs stained with ethidium bromide were used as loading controls. A set of ³²P-labeled RNA oligos were electrophoresed in parallel and used as size markers. The blots were stripped and re-probed. See Figures S1, Table S1 and Table S2 for additional information about sidRNA identification in seedling, Figure S2, Figure S3 and Table S4 for information about detection of sidRNAs in other systems.

Figure 2. Effects of Pol IV/RDR2 Mutations on sidRNA Production

(A) Venn diagram showing the overlap between sidRNA loci in dcl1/2/3/4 and P4siRNA loci in Col-0. (B) Pie chart showing the numbers of sidRNA loci dependent on NRPD1 and/or RDR2. (C) Heat map of the abundance of sRNAs generated from sidRNA loci in the indicated plants. (D) Pie chart summarizing the numbers of NRPD1/RDR2-dependent and -independent sidRNA loci in the indicated categories. (E) Detection of sRNA production at representative sidRNA loci in the indicated plants by Northern blot. 5S rRNAs stained with ethidium bromide were used as loading controls. (F) Box plots showing the strand bias of sidRNAs in dcl2/3/4 and nrpd1 dcl2/3/4 mutants. Strand bias value was calculated as the absolute (abs) percentage differences between sRNAs reads produced from the positive (pos) and negative (neg) strands of sidRNA loci. See also Table S3.

Figure 3. sidRNAs Are Associated with AGO4

(A) Heatmap showing the size distribution of AGO4-associated sRNAs from sidRNA-producing loci in Col-0, dcl2/3/4 and dcl1/2/3/4. Color intensity represents the fraction of sRNAs of different sizes. (B) Box plots of the percentages of laddered AGO4-associated sRNAs produced from sidRNA loci in Col-0 and dcl2/3/4. (C) A representative group of laddered AGO4-associated sRNAs produced from AtREP2. Numbers represent the abundance (RPM) of sidRNAs with different lengths ranging from 19 to 29 nt. Values in the brackets indicate the percentages. (D) Detection of sRNAs at representative sidRNA loci in total extracts and AGO4 immunoprecipitates by Northern blot. 5S rRNAs stained with ethidium bromide were used as loading controls for total extracts. A silver-stained gel shows that comparable amounts of AGO4 immunoprecipitates were used for sRNA extraction.

Figure 4. sidRNAs Are Capable of Directing DNA Methylation

(A) Detection of AGO4 occupancy at the indicated loci in Col-0 and the indicated mutants by ChIP analysis. AGO4 ChIP signals in the mutants are presented in relation to those in Col-0 (arbitrarily set to 1.0). Error bars indicate standard deviations of three biological replicates. Asterisks indicate a significant difference between the indicated groups (P < 0.05, t test ). (B) Analysis of DNA methylation at the indicated loci in Col-0 and the indicated mutants by bisulfite sequencing. Presented is overall percent methylation of cytosine sites in different sequence contexts. More than 20 clones were sequenced for each sample (C) Box plots of CG, CHG and CHH methylation levels at sidRNA loci in Col-0 and the indicated mutants. Asterisks indicate a significant difference between the indicated groups (P < 10⁻¹⁵, Mann–Whitney U test). (D) Heat map of CG, CHG and CHH methylation levels at sidRNA loci in Col-0 and the indicated mutants. See also Table S5 and Table S6.

Figure 5. Probing The Mechanism Underlying sidRNA Biogenesis

(A) A model for sidRNA maturation. A sidRNA precursor transcript is bound by AGO4 and subsequently trimmed from the 3’-end by a 3’-5’ distributive exonuclease to yield a mature sidRNA. (B) Box plots of the abundance of sidRNAs in the indicated plants. Asterisks indicate a significant difference between the indicated groups (P < 10⁻¹⁵, Mann–Whitney U test). (C) Detection of sRNA production at representative sidRNA loci in the indicated plants by Northern blot. 5S rRNAs stained with ethidium bromide were used as loading controls. (D) Sensitivity of sidRNAs to Terminator exonuclease. sRNAs in total extracts and AGO4 immunoprecipitates prepared from Col-0 and dcl2/3/4 were treated with Terminator exonuclease and AtREP2 sRNAs were probed by Northern blot. A 21-nt spike-in RNA with 5’ monophosphate was added in the reactions and used as a positive control. (E) Sensitivity of sidRNAs to RNase T1. sRNAs in total extracts and AGO4 immunoprecipitates prepared from Col-0 and dcl2/3/4 were treated with RNase T1 and AtREP2 sRNAs were probed by Northern blot. Spike-in RNAs including a 50-bp double-stranded RNA with 2-nt 3’ over-hang (ds) and a 50-nt single-stranded (ss) RNA were added in the reactions and used as controls for RNase T1 digestion.

Figure 6. Distributive 3’-5’ Exonucleases Are Required for siRNA Accumulation and DNA Methylation

(A) Identification of distributive 3’-5’ exonucleases required for DNA methylation. Diagrams show the structure of Atrimmer1/RRP6L1 and Atrimmer2 (Upper panel). The positions of residues forming the conserved DEDD-Y active site are indicated. The bottom panel shows DNA methylation levels at the AtSN1 and IGN5 loci in the indicated plants as measured by Chop-PCR. The methylation-sensitive restriction enzyme HaeIII was used to specifically cleave unmethylated DNA in CHH context. A fragment of Actin that lacks HaeIII sites was amplified as a control. See Figure S4 for Chop-PCR results from all tested 3’-5’ exonuclease mutants. (B) Venn diagram showing the numbers of sidRNA loci affected by atrimmer1/rrp6l1, atrimmer2 or both. (C) Box plots of the levels of sidRNAs generated from Atrimmer1- (Left panel) and Atrimmer2- (Right panel) affected sidRNA loci in the indicated plants. (D) Relative levels (shown in RPMs) of representative Atrimmer1/2-controlled sidRNA loci in the indicated plants measured by deep sequencing. (E) Detection of sRNA production at representative sidRNA loci in the indicated plants by Northern blot. 5S rRNAs stained with ethidium bromide were used as loading controls. (F) Analysis of DNA methylation at the indicated loci in Col-0 and the indicated mutants by bisulfite sequencing. Presented is overall percent methylation of cytosine sites in different sequence contexts. More than 20 clones were sequenced for each sample. See also Figure S4 and Table S7.

Figure 7. A Model for Biogenesis of sidRNAs and Initiation of DNA Methylation

In this model, an active locus (for example, a newly integrated transgene or transposon) is transcribed by Pol II. Pol II transcripts (possibly amplified by RDR6). associate with AGO4 and undergo 3’-5’ trimming by Atrimmers. The resulting sidRNAs then mediate the initiation of DNA methylation by recruiting DRM2. Low levels of DNA methylation established by sidRNAs may mark the locus for subsequent recruitment of Pol IV and RDR2. The vast majority of the Pol IV/RDR2 transcripts are processed by DCL3 to generate 24-nt siRNAs that are subsequently loaded onto AGO4, while a small fraction of them are directly recruited by AGO4 and undergo 3’-5 trimming to produce sidRNAs. 24-nt siRNAs and sidRNAs then recruit DRM2 by base pairing with scaffold transcripts generated by Pol V, to reinforce DNA methylation.