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. Overall design: 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

由24核苷酸（nt）小干扰RNA（small interfering RNAs, siRNAs）介导的DNA甲基化（DNA methylation），在拟南芥的基因调控与转座子沉默过程中发挥关键作用。已知24 nt siRNA由双链RNA经Dicer样蛋白3（Dicer-like 3, DCL3）切割产生，并加载至效应蛋白Argonaute 4（AGO4）中。本研究报道了一类不依赖DCL家族的独特小RNA（sidRNAs）：这类sidRNA以长度约20至60 nt的阶梯状分布形式存在，通常具有一致的5'端，而3'端以1 nt的步长呈现差异。本研究进一步证实，sidRNA可与AGO4结合，并能够介导DNA甲基化。此外，研究发现sidRNA的生成依赖于AGO4以及分布式3'-5'核酸外切酶。本研究结果揭示了一条全新的siRNA生物合成途径：前体转录本先与AGO4结合，随后经3'-5'外切酶的降解修剪以完成成熟过程。我们推测，通过该途径生成的sidRNA是从头DNA甲基化（de novo DNA methylation）的初始触发因子。实验设计概述：通过Illumina高通量测序，对拟南芥野生型（Col-0）、dcl1/2/3/4突变体及其他相关突变体中的小RNA进行测序分析，以鉴定不依赖DCL家族的siRNA（sidRNAs）并解析其生物合成途径；同时，通过全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）对多种突变体的DNA甲基化水平进行检测，以验证sidRNA在DNA甲基化中的功能。