Dissection of the piRNA biogenesis pathway in germ cells

项目摘要和摘要 以Piwi蛋白为中心的小分子调控RNA通路及其结合 Piwi相互作用RNA(PiRNAs)沉默转座子和其他靶点以维持动物基因组的完整性 生殖系。干扰piRNA途径导致转座子水平升高和生殖系缺陷 发育，最终导致动物不育。因此，piRNA途径起到了守护者的作用 生殖系基因组，以确保遗传信息传递给下一代。尽管至关重要的是 PiRNA在生殖系发育中的作用是显而易见的，介导piRNA生物发生的因素和机制 仍然难以捉摸。PiRNAs的生物发生始于转录定义的长单链RNA 称为piRNA簇的基因组区域。前体转录很可能被处理成较短的初级 先驱物。然而，初级piRNA前体的加工概况和机制并不是很好 已澄清。初级前体由线粒体相关酶西葫芦进一步处理，以 产生Pre-piRNAs，它被加载到Piwi蛋白上，在那里它们经历3‘端加工和 甲基化成为成熟的piRNA。为了阐明piRNAs的生物发生途径， 我们一直在利用家蚕卵巢来源的BmN4细胞，它内源性地表达Piwi蛋白和 PIRNA。表达piRNA的可培养细胞的稀缺使BmN4细胞适合并很好地用于piRNA 生物发生学研究。在细胞中，由tRNAs大量产生piRNAs，我们最近揭示了 TRNA衍生的piRNAs的生物发生机制，其中5‘-tRNA成为直接的，而不是成熟的tRNAs PiRNA的前体。我们在5‘-tRNA半链的3’-末端发现了2‘，3’-环磷酸(CP)，这促使我们 假设含CP的RNA(CP-RNAs)形成piRNA前体。我们的假设得到了强化 我们的进一步分析表明，在BmN4细胞中，CP-RNAs在细胞外大量积累 发生piRNA生物发生的线粒体膜，这些CP-RNA序列是 与piRNA序列广泛重叠。CP-RNA形成转录组的隐藏层，因为 标准的rna-seq无法捕捉到它们。我们开发了一种CP-RNA-SEQ方法，它可以选择性地 测序CP-RNAs，并利用它对家蚕BmN4中表达的CP-RNAs进行综合鉴定 细胞、果蝇Kc167细胞和小鼠睾丸，以探讨它们与 PiRNAs(目标1)。我们将进一步阐明CP-RNAs与Piwi蛋白的相互作用和PiRNA的生物发生 因子BmPapi(目标2)，并鉴定在piRNA生物发生中产生CP-RNAs的RNase(目标3)。这个 拟议的研究将推动我们的新研究努力，通过以下方式了解piRNA的生物发生机制 关注目前尚未确定的CP-RNAs，并支持生物医学目标以了解发病机制 生殖系统疾病和异位表达Piwi蛋白的癌症。

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.