Genetic and mechanistic diversity of piRNA 3' end formation. Drosophila melanogaster

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI- interacting RNAs (piRNAs)—the 22–30-nt-long guides for PIWI- clade Ago proteins that silence transposons in animal gonads— are generated independently of Dicer from single-stranded precursors. piRNA 5′ ends are defined either by Zucchini, the Drosophila homologue of mitoPLD—a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3′ ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3′ ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini- mediated cleavages directly define mature piRNA 3′ ends, Aub/ Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3′-to-5′ exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5′ and 3′ ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3′-end formation. Overall design: This study aims at understanding how piRNA 3' ends are formed in Drosophila and what the role of the exonuclease Nibbler is in their formation.

小型调控RNA（small regulatory RNAs）以序列特异性方式引导Argonaute蛋白（Ago）靶向其作用靶点，因此在真核基因沉默过程中发挥关键作用。在三类小RNA中，微小RNA（microRNAs）与小干扰RNA（short interfering RNAs）均由Dicer——一种具有固有标尺功能的核糖核酸内切酶——从双链前体加工为定型的21~23 nt片段。PIWI相互作用RNA（PIWI-interacting RNAs, piRNAs）是长度为22~30 nt的向导RNA，可介导PIWI进化枝Ago蛋白沉默动物性腺中的转座子，其生成不依赖Dicer，且源自单链前体。piRNA的5'端可通过两种方式定义：一是Zucchini蛋白，即线粒体锚定核糖核酸内切酶mitoPLD的果蝇同源物；二是piRNA引导的靶标切割。目前学界对piRNA 3'端的形成机制尚缺乏深入了解。本研究报道，黑腹果蝇中存在两条遗传与机制均截然不同的通路，可分别介导piRNA 3'端的生成。两条通路的起始核酸酶分别为Zucchini蛋白，或是PIWI进化枝蛋白Aubergine（Aub）/Ago3。Zucchini介导的切割可直接确定成熟piRNA的3'端，而Aub/Ago3介导的切割则释放前体piRNA，此类前体需要借助3'→5'外核糖核酸酶Nibbler（果蝇Mut-7同源物）进行广泛的末端切除。两条通路的相对活性决定了piRNA被导向细胞质还是细胞核PIWI进化枝蛋白的比例，进而调控转录后沉默与转录沉默之间的动态平衡。值得注意的是，同时缺失Zucchini与Nibbler的实验体系揭示了一条极简的、由Argonaute驱动的小RNA生成通路：piRNA的5'与3'端可由紧密间隔的Aub/Ago3介导的切割事件直接产生。本研究的数据阐明了一套连贯的piRNA生成模型，将有助于解析调控piRNA 3'端形成的具体分子机制。整体实验设计：本研究旨在阐明黑腹果蝇中piRNA 3'端的形成机制，以及外核糖核酸酶Nibbler在该过程中的具体功能。