Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Curr Biol. 2011 Mar 22;21(6):449-59. doi: 10.1016/j.cub.2011.02.019.
The development of the germline in Caenorhabditis elegans is a complex process involving the regulation of thousands of genes in a coordinated manner. Several genes required for small RNA biogenesis and function are among those required for the proper organization of the germline. EGO-1 is a putative RNA-directed RNA polymerase (RdRP) that is required for multiple aspects of C. elegans germline development and efficient RNA interference (RNAi) of germline-expressed genes. RdRPs have been proposed to act through a variety of mechanisms, including the posttranscriptional targeting of specific mRNAs, as well as through a direct interaction with chromatin. Despite extensive investigation, the molecular role of EGO-1 has remained enigmatic.
Here we use high-throughput small RNA and messenger RNA sequencing to investigate EGO-1 function. We found that EGO-1 is required to produce a distinct pool of small RNAs antisense to a number of germline-expressed mRNAs through several developmental stages. These potential mRNA targets fall into distinct classes, including genes required for kinetochore and nuclear pore assembly, histone-modifying activities, and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong association between the loss of small RNAs and the rise of mRNA levels in ego-1(-) animals.
Our data support the conclusion that EGO-1 produces triphosphorylated small RNAs derived from mRNA templates and that these small RNAs modulate gene expression through the targeting of their cognate mRNAs.
秀丽隐杆线虫生殖系的发育是一个复杂的过程，涉及数千个基因的协调调控。在生殖系的正常组织中，有几个需要的基因参与小 RNA 的生物发生和功能。EGO-1 是一种假定的 RNA 指导的 RNA 聚合酶（RdRP），它需要多个方面的秀丽隐杆线虫生殖系发育和有效的生殖系表达基因的 RNA 干扰（RNAi）。RdRPs 被提议通过多种机制发挥作用，包括对特定 mRNA 的转录后靶向，以及与染色质的直接相互作用。尽管进行了广泛的研究，但 EGO-1 的分子作用仍然是一个谜。
在这里，我们使用高通量小 RNA 和信使 RNA 测序来研究 EGO-1 的功能。我们发现 EGO-1 在几个发育阶段都需要产生一种独特的小 RNA 库，这些小 RNA 与许多生殖系表达的 mRNA 反义。这些潜在的 mRNA 靶标分为不同的类别，包括动粒和核孔组装、组蛋白修饰活性和着丝粒蛋白所需的基因。我们还发现几个 RNAi 相关基因是 EGO-1 的靶标。最后，我们发现 ego-1(-) 动物中小 RNA 的缺失与 mRNA 水平的升高之间存在很强的关联。
我们的数据支持这样的结论，即 EGO-1 产生来源于 mRNA 模板的三磷酸化小 RNA，这些小 RNA 通过靶向其同源 mRNA 来调节基因表达。
