Talmor-Neiman Mali, Stav Ran, Klipcan Liron, Buxdorf Kobi, Baulcombe David C, Arazi Tzahi
Department of Ornamental Horticulture, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel.
Plant J. 2006 Nov;48(4):511-21. doi: 10.1111/j.1365-313X.2006.02895.x.
Trans-acting small interfering RNAs (tasiRNAs) are a class of higher-plant endogenous siRNAs that, like miRNAs, direct the cleavage of non-identical transcripts. tasiRNAs derive from non-coding transcripts (TAS) that are converted into dsRNA by a RNA-dependent RNA polymerase (RDR6), following their initial miRNA-guided cleavage. The dsRNA is then processed by a dicer-like enzyme 4 into phased 21-nucleotide siRNAs. To date, tasiRNAs have been identified only in Arabidopsis, and their identity and function in other land plants are unknown. Here, a set of endogenous small RNAs that correspond in a phased manner to a non-coding transcript (contig13502) were identified in the moss Pyscomitrella patens. Northern analysis suggests that contig13502-derived small RNAs are expressed in the juvenile gametophyte. In addition, miR390-guided cleavage of contig13502 at two sites flanking the small RNAs cluster was validated by 5' RACE. These cleavages are predicted to provide defined termini for the production of phased siRNAs. To elucidate the biogenesis of identified siRNAs, we cloned and generated knock-out mutants for an RDR6 moss homologue (PpRDR6). These mutants exhibited an accelerated transition from juvenile to mature gametophyte. In addition, RNA blots demonstrated that they lacked contig13502-derived siRNAs, suggesting that PpRDR6 is required for siRNA biogenesis. A target gene, which showed homology to an AP2/EREBP transcription factor, for one phased siRNA, was validated, corroborating its identity as a trans-acting siRNA. Taken together, our data indicate that contig13502 is a novel TAS locus and suggest a role for derived tasiRNAs in the regulation of gene expression in moss.
反式作用小干扰RNA（tasiRNA）是一类高等植物内源性小干扰RNA，与微小RNA（miRNA）一样，指导非同源转录本的切割。tasiRNA来源于非编码转录本（TAS），在最初的miRNA引导切割后，由RNA依赖性RNA聚合酶（RDR6）将其转化为双链RNA（dsRNA）。然后，双链RNA由类Dicer酶4加工成21个核苷酸的阶段性小干扰RNA。迄今为止，仅在拟南芥中鉴定出tasiRNA，其在其他陆地植物中的特性和功能尚不清楚。在此，在小立碗藓中鉴定出一组与非编码转录本（重叠群13502）呈阶段性对应关系的内源性小RNA。Northern分析表明，来源于重叠群13502的小RNA在幼年配子体中表达。此外，通过5'RACE验证了miR390在小RNA簇两侧的两个位点对重叠群13502的切割。预计这些切割将为阶段性小干扰RNA的产生提供确定的末端。为了阐明已鉴定小干扰RNA的生物合成，我们克隆并生成了RDR6苔藓同源物（PpRDR6）的敲除突变体。这些突变体表现出从幼年到成熟配子体的加速转变。此外，RNA印迹表明它们缺乏来源于重叠群13502的小干扰RNA，这表明PpRDR6是小干扰RNA生物合成所必需的。一个阶段性小干扰RNA的靶基因被验证，该基因与AP2/EREBP转录因子具有同源性，证实了其作为反式作用小干扰RNA的特性。综上所述，我们的数据表明重叠群13502是一个新的TAS位点，并暗示衍生的tasiRNA在苔藓基因表达调控中发挥作用。
