Dicing of double-stranded RNA (dsRNA) into small RNA is an essential process to trigger transcriptional and post-transcriptional gene silencing. Using cell-free extracts of the model filamentous fungus Neurospora crassa, we successfully detected the dicing activity of one of two N. crassa Dicers NcDCL2. The predominant 23-nucleotide (nt) cleavage product was always detected from 30-nt to 130-nt dsRNA substrates, and additional products of approximately 18 to 28 nt were occasionally produced. The enzymatic properties of NcDCL2 are different from those of insect and plant small interfering RNA (siRNA)-producing Dicers, Drosophila melanogaster Dicer-2 and Arabidopsis thaliana DCL3 and DCL4 (AtDCL3 and AtDCL4). Whereas AtDCL3 and AtDCL4 preferentially cleave short and long dsRNAs, respectively, NcDCL2 cleaved both short and long dsRNAs. These results suggest that N. crassa has a single siRNA-producing Dicer NcDCL2, which is a prototype of plant siRNA-producing Dicers with distinct functions in diverse RNA silencing pathways. The dicing assay reported here is convenient to detect and biochemically characterize the dicing activities of both plant and fungal Dicers, and is likely applicable to other organisms.

将双链 RNA (dsRNA) 切割成小 RNA 是触发转录和转录后基因沉默的重要过程。使用模型丝状真菌粗糙脉孢菌的无细胞提取物，我们成功地检测到两个粗糙脉孢菌Dicer NcDCL2之一的切割活性。从 30-nt 到 130-nt dsRNA 底物总是检测到主要的 23 核苷酸 (nt) 切割产物，偶尔会产生大约 18 到 28 nt 的额外产物。NcDCL2 的酶学性质不同于昆虫和植物产生小干扰 RNA (siRNA) 的 Dicer、Drosophila melanogaster Dicer-2 和拟南芥DCL3 和 DCL4（AtDCL3 和 AtDCL4）。AtDCL3 和 AtDCL4 分别优先切割短dsRNA 和长dsRNA，而NcDCL2 切割短dsRNA 和长dsRNA。这些结果表明，粗糙脉孢菌具有单一的产生 siRNA 的 Dicer NcDCL2，它是植物产生 siRNA 的 Dicer 的原型，在不同的 RNA 沉默途径中具有不同的功能。此处报告的切割试验便于检测和生化表征植物和真菌 Dicer 的切割活性，并且可能适用于其他生物。