Plant 22-nt siRNAs mediate translational repression and stress adaptation

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, ein5-1, ski2-2, dcl4-2, dcl2-1, ein5-1 dcl4-2, ein5-1 dcl2-1, ein5-1 dcl4-2 dcl2-1, ski2-2 dcl4-2, ski2-2 dcl2-1 and ski2-2 dcl4-2 dcl2-1 were grown on the MS medium for 6 days and then were transferred to the soil for another 14 days. Aerial parts of 20-day-old plant were collected at 15:00 for total 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 RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). Sequencing libraries were prepared using the TruSeq mRNA preparation kit (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 Illumina HiSeqX ten platform.

小干扰RNA（small interfering RNAs, siRNAs）对真核生物的正常发育与免疫至关重要¹。植物可产生长度为21、22或24核苷酸（nucleotides, nt）的siRNA，其中21nt与24nt的siRNA分别介导mRNA剪切与DNA甲基化²,³。然而，22nt siRNA的生物学功能仍尚不明确。本研究鉴定并表征了一类由类Dicer蛋白2（DICER-LIKE 2, DCL2）催化生成的内源性22nt siRNA。当细胞质RNA降解途径缺陷且DCL4功能缺失时，大量积累的22nt siRNA会引发多效性生长紊乱，包括严重矮化、分生组织缺陷与色素沉着异常。值得注意的是，编码硝酸还原酶的两个基因NIA1与NIA2所产生的22nt siRNA占总22nt siRNA总量的近一半。22nt siRNA的生成可触发爆发式自我扩增，形成小RNA风暴，并同时引发基因特异性与全局性的显著翻译抑制。此外，研究发现22nt siRNA在氮缺乏条件下会优先积累，进而抑制植物生长并促进胁迫响应。综上，本研究揭示了此前未被探究的一类植物siRNA——22nt siRNA的独特性质，并强调了小RNA长度是其核心功能决定因子。
本研究将Col-0、ein5-1、ski2-2、dcl4-2、dcl2-1、ein5-1 dcl4-2、ein5-1 dcl2-1、ein5-1 dcl4-2 dcl2-1、ski2-2 dcl4-2、ski2-2 dcl2-1与ski2-2 dcl4-2 dcl2-1共11种基因型的植株接种于MS培养基培养6天，随后移栽至土壤中继续生长14天。于每日15:00收集20日龄植株的地上部分，用于总RNA提取。每种基因型设置3次生物学重复。建库与高通量测序均按照试剂盒厂商说明书进行。总RNA提取采用RNeasy植物微量提取试剂盒（RNeasy Plant Mini Kit, Qiagen）；测序文库构建使用TruSeq mRNA建库试剂盒（TruSeq mRNA preparation kit, Illumina）。RNA质量与文库质量通过2100生物分析仪（Bioanalyzer 2100 instrument, Agilent）检测，随后在Illumina HiSeqX十通量测序平台上完成150bp双端深度测序。