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

DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-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 to 60nt in length, often having 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 AGO4 and 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. Small RNAs were profiled in Arabidopsis wild type (Col-0), dcl1/2/3/4 and other mutants by Illumina high-throughput sequencing, to identify siRNAs independent of DCLs (sidRNAs) and dissect their biogenesis pathway. DNA methylation was profiled in various mutants by whole-genome bisulfite sequencing to establish a role for sidRNAs in DNA methylation