State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Plant J. 2012 Feb;69(3):462-74. doi: 10.1111/j.1365-313X.2011.04805.x. Epub 2011 Nov 23.
Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.
高等植物进化出多种 RNase III 家族蛋白，以产生和调控具有特殊分子功能的不同小 RNA 类别。在水稻（Oryza sativa）中，有两个 microRNA（miRNA），miR2118 和 miR2275，靶向许多基因组簇，以产生 21 或 24 个核苷酸的二级小干扰 RNA（siRNA），呈阶段性排列。这些阶段性小 RNA 的生物发生要求或功能完全未知。在这里，我们研究了水稻 Dicer-like（DCL）家族，包括 OsDCL1、-3a、-3b 和 -4。通过对野生型和 osdcl4-1 不同组织的小 RNA 进行深度测序，我们揭示了包括转座 siRNA（tasiRNA）和 1000 多个阶段性小 RNA 基因座在内的 21 个核苷酸 siRNA 的加工过程在很大程度上依赖于 OsDCL4。令人惊讶的是，24 个核苷酸阶段性小 RNA 的加工需要 DCL3 同源物 OsDCL3b，而不是 OsDCL3a，这表明 DCL3 家族内存在功能分化。RNA 连接酶介导的 5' 快速扩增 cDNA 末端和并行分析 RNA 末端（PARE）/降解组分析证实，大多数 21 和 24 个核苷酸阶段性小 RNA 簇分别从 miR2118 和 miR2275 的靶位点起始。此外，两种触发 miRNA 的积累需要 OsDCL1 的活性。最后，我们表明阶段性小 RNA 优先在雄性生殖器官中产生，并且可能在单子叶植物中保守。我们的研究结果揭示了 OsDCL4、OsDCL3b 和 OsDCL1 在水稻 21 和 24 个核苷酸阶段性小 RNA 生物发生途径中的重要作用。
