Patrick Kristin L, Shi Huafang, Kolev Nikolay G, Ersfeld Klaus, Tschudi Christian, Ullu Elisabetta
Department of Epidemiology and Public Health, Yale University Medical School, 295 Congress Avenue, New Haven, CT 06536-0812, USA.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17933-8. doi: 10.1073/pnas.0907766106. Epub 2009 Oct 6.
Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.
布氏锥虫是最古老的真核生物之一，其RNA干扰（RNAi）机制可正常运行，也是唯一一种RNAi得到广泛研究并被用作功能分析工具的单细胞病原体。在此，我们报告称，布氏锥虫的RNAi途径虽然依赖单一的AGO1蛋白，但由两种不同的类Dicer酶的活性启动。主要定位于细胞质的TbDCL1和之前未描述过的核酶TbDCL2都参与了反转录转座子来源的小干扰RNA（siRNA）的生物合成。然而，TbDCL2在从染色体内部重复转录本产生siRNA方面起主要作用，这些转录本在RNAi缺陷细胞的核仁中积累，并启动针对反转录转座子和重复序列的内源性RNAi反应。此外，由TbDCL1和TbDCL2产生的siRNA都带有5'-单磷酸和封闭的3'末端，这表明3'末端修饰是siRNA的一个古老特征。因此，我们提出了一个模型，即TbDCL2推动布氏锥虫的核RNAi途径，而TbDCL1负责巡查细胞质，在转录后沉默可能进入细胞质的潜在有害核酸寄生虫。不过，我们也提供了两种类Dicer酶之间存在相互作用的证据，因为TbDCL2与35至65个核苷酸的中间转录本的产生有关，这些转录本似乎是TbDCL1的底物。我们发现dcl2基因敲除细胞比野生型细胞对RNAi触发物更敏感，这对这种重要的人类病原体的反向遗传学分析具有重要意义。
