Function, targets and evolution of Caenorhabditis elegans piRNAs (small RNA-Seq)

piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. Overall design: 7 small RNA libraries were sequenced as part of 25 flow cell lanes on the Illumina GA II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNAs with a 5'-triphosphate. Samples were labelled for multiplexing using 4-bp 5'-barcodes or barcodes included in Illumina TruSeq adapters. In most cases a single flow cell lane included several multiplexed libraries.

PIWI互作RNA（piRNAs, Piwi-interacting RNAs）对于维持生殖系完整性与生育能力不可或缺，但其作用机制仍未被充分阐明。本研究证实，秀丽隐杆线虫（C. elegans, Caenorhabditis elegans）的piRNAs可通过不完全互补位点反式沉默转录本。研究发现，靶标沉默并不依赖于Piwi的核酸内切酶活性或‘切割（slicing）’功能。取而代之的是，本研究表明piRNAs可触发局域性次级内源性小干扰RNA（endo-siRNA）应答反应。内源性蛋白编码基因、假基因以及转座子的转录本在与piRNAs互补的位点上呈现Piwi依赖型endo-siRNA表达，并在Piwi突变体中发生去抑制。piRNA生成的基因组位点中蛋白编码基因匮乏，但假基因与转座子并不受此限制。本研究数据表明，线虫piRNA簇正处于演化进程中，以生成靶向活性移动遗传元件的piRNAs。因此，在秀丽隐杆线虫中，piRNAs可作为可遗传的序列特异性RNA干扰（RNAi, RNA interference）触发因子。实验设计概述：本研究在Illumina GA II测序平台的25个流动槽泳道中完成了7个小RNA文库的测序。样本经烟草酸焦磷酸酶处理，以实现对带有5'-三磷酸基团的小RNA的克隆。样本通过4碱基5'端条形码或Illumina TruSeq接头内置条形码进行标记，以实现多重测序。多数情况下，单个流动槽泳道可包含多个多重化文库。