Dissection of the piRNA biogenesis pathway in germ cells

项目概要和摘要 Piwi（P-element-induced wimpy testis）蛋白及其结合的小调控RNA途径 Piwi相互作用RNA（piRNA）沉默转座子和其他靶标以维持动物中的基因组完整性 生殖系皮尔纳途径的破坏导致转座子水平的升高和生殖系中的缺陷 发育，最终导致动物不育。因此，皮尔纳途径充当以下物质的监护人： 生殖细胞基因组，以确保遗传信息传递给下一代。虽然关键的 piRNA在种系发育中的功能是明显的，介导皮尔纳生物发生的因素和机制 仍然难以捉摸piRNA的生物发生开始于从限定的DNA序列转录长单链RNA。 称为皮尔纳簇的基因组区域。前体转录本可能被加工成较短的初级转录本。 前体然而，初级皮尔纳前体的加工概况和机制尚不清楚。 阐明。初级前体被与西葫芦相关的酶进一步加工， 产生pre-piRNA，其被加载到Piwi蛋白上，在那里它们经历3′端加工， 甲基化以成为成熟的piRNA。为了我们的研究目标，以澄清piRNA的生物合成途径， 我们一直在利用来源于家蚕卵巢的BmN 4细胞，其内源性表达Piwi蛋白， piRNA。表达piRNA的可培养细胞的稀缺性使得BmN 4细胞适合并良好地用于皮尔纳。 生物起源研究在细胞中，piRNA大量地由tRNA产生，我们最近揭示了 tRNA衍生的piRNA的生物发生机制，其中5′-tRNA半体，而不是成熟的tRNA， piRNA的前体。我们在5′-tRNA的3′-末端发现了一个2′，3 ′-环磷酸（cP），这促使我们 假设含cP的RNA（cP-RNA）形成皮尔纳前体。我们的假设得到了加强 通过我们进一步的分析表明，在BmN 4细胞中，cP-RNA在细胞外表面大量积累， 线粒体的膜，其中皮尔纳生物发生，并且这些cP-RNA序列是 与皮尔纳序列广泛重叠。cP-RNA形成转录组的隐藏层， 标准RNA-seq无法捕获它们。我们开发了一种cP-RNA-seq方法， 测序cP-RNA，我们将利用它来全面鉴定在BmN 4中表达的cP-RNA 细胞，果蝇Kc 167细胞和小鼠睾丸，以研究它们的表达关系， piRNA（Aim 1）。我们将进一步阐明cP-RNA与Piwi蛋白的相互作用以及皮尔纳的生物发生。 因子BmPapi（Aim 2），并表征皮尔纳生物发生中产生cP-RNA的RNA酶（Aim 3）。的 拟议的研究将推动我们的新的研究工作，以了解皮尔纳生物合成的机制， 专注于目前未表征的cP-RNA，并支持生物医学目标，以了解发病机制 异位表达Piwi蛋白的生殖系统疾病和癌症。

Project Summary and Abstract The small regulatory RNA pathway centered on Piwi (P-element-induced wimpy testis) proteins and their bound Piwi-interacting RNAs (piRNAs) silence transposons and other targets to maintain genome integrity in animal germlines. Disruption of the piRNA pathway causes elevation of transposon levels and defects in germline development, eventually resulting in sterility of the animals. The piRNA pathway thus functions as guardians of the germline genome to ensure genetic information is passed onto the next generation. Although the crucial function of piRNAs in germline development is evident, the factors and mechanisms mediating piRNA biogenesis remain elusive. Biogenesis of the piRNAs starts by transcription of long single-stranded RNAs from defined genomic regions termed piRNA clusters. Precursors transcripts are likely processed into shorter primary precursors. However, profiles and mechanisms of the processing for primary piRNA precursors are not well elucidated. The primary precursors are further processed by the mitochondria-associated enzyme Zucchini to generate pre-piRNAs, which are loaded onto Piwi proteins where they undergo 3′-end processing and methylation to become mature piRNAs. Toward our research goal to clarify the biogenesis pathway of piRNAs, we have been utilizing Bombyx mori ovary-derived BmN4 cells which endogenously express Piwi proteins and piRNAs. The scarcity of piRNA-expressing culturable cells make BmN4 cells suitable and well used for piRNA biogenesis research. In the cells, piRNAs are produced abundantly from tRNAs, and we recently revealed the biogenesis mechanism of the tRNA-derived piRNAs, in which 5′-tRNA halves, not mature tRNAs, become direct precursors for piRNAs. Our finding of a 2′,3′-cyclic phosphate (cP) at the 3′-end of 5′-tRNA halves prompted us to hypothesize that cP-containing RNAs (cP-RNAs) form piRNA precursors. Our hypothesis was strengthened by our further analyses showing that, in BmN4 cells, cP-RNAs are abundantly accumulated at the outer membrane of mitochondria where piRNA biogenesis takes place, and that these cP-RNA sequences are extensively overlapped with piRNA sequences. cP-RNAs form a hidden layer of the transcriptome because standard RNA-seq is unable to capture them. We have developed a cP-RNA-seq method that can selectively sequence cP-RNAs, and we will utilize it to comprehensively identify the cP-RNAs expressed in Bombyx BmN4 cells, Drosophila Kc167 cells, and mouse testes in order to investigate their expressional relationship with piRNAs (Aim 1). We will further elucidate the interaction of cP-RNAs with Piwi proteins and a piRNA biogenesis factor BmPapi (Aim 2), and characterize an RNase producing cP-RNAs in piRNA biogenesis (Aim 3). The proposed studies will fuel our novel research efforts to understand the mechanism of piRNA biogenesis by focusing on currently-uncharacterized cP-RNAs, and support biomedical goals to understand the pathogenesis of reproductive system diseases and cancers that ectopically express Piwi proteins.