Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals novel siRNA loci

Summary Plant small RNAs regulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. sRNAs fall into two major categories: those that are reliant on RNA Dependent RNA Polymerases ( RDRs ) for biogenesis and those that aren’t. Known RDR -dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR -independent sRNAs are primarily microRNAs and other hairpin-derived sRNAs. In this study, we produced and analyzed small RNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. Only a small fraction of all sRNA loci were RDR1/RDR2/RDR6 -independent; most of these were microRNA loci or associated with predicted hairpin precursors. We found 58 previously annotated microRNA loci that were reliant on RDR1 , − 2 , or − 6 function, casting doubt on their classification. We also found 38 RDR1/2/6 -independent small RNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for small RNA biogenesis. These 38 small RNA-producing loci have novel biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggest that these 38 loci represent one or more new types of small RNAs in Arabidopsis thaliana . Significance Statement Small RNAs regulate gene expression in plants and are produced through a variety of previously-described mechanisms. Here, we examine a set of previously undiscovered small RNA-producing loci that are produced by novel mechanisms.