22-nucleotide siRNAs mediate translational repression and trigger a silencing storm

Small interfering RNAs (siRNAs) are critical for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21-, 22-, or 24- nucleotides (nt), wherein the 21- and 24-nt siRNAs mediate mRNA cleavage and DNA methylation2,3, respectively. However, the biological functions of 22-nt siRNAs remain elusive. Here we report the identification and characterization of a group of endogenous 22-nt siRNAs generated from the action of DICER-LIKE 2 (DCL2). When cytoplasmic RNA decay and DCL4 are deficient, the massive accumulation of 22-nt siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defect, and pigmentation. Notably, two genes that encode nitrate reductases, NIA1 and NIA2, produce nearly half of the total of 22-nt siRNAs. Production of 22-nt siRNA triggers explosive self-amplification that leads to a small RNA storm, and induces dramatic translational repression both gene-specifically and globally. 22-nt siRNAs are also found to preferentially accumulate upon nitrogen deficiency, which acts to restrain plant growth and promote stress responses. Thus, our research uncovers the unique properties of 22-nt siRNAs, a previously unexplored class of plant siRNAs, and highlights the length of small RNA as a major functional determinant. Col-0, dcl4-2 and dcl4-2 dcl2-1 were grown on the MS or 1μM ABA medium for 14 days and seedlings were collected for small RNA extraction. Three biological replicates were prepared for each genotype. Library preparation and high-throughput sequencing were conducted in accordance with the manufacturers’ instructions. Total small RNA was extracted using the miRNeasy Mini Kit (Qiagen). Sequencing libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina. RNA quality and library quality were examined by a Bioanalyzer 2100 instrument (Agilent), and paired-end, 150-bp deep sequencing was performed on an Noveseq6000 platform. Only read1 was used for downstream analysis