Dissection of the piRNA biogenesis pathway in germ cells

生殖细胞中 piRNA 生物发生途径的剖析

• 批准号: 10158519
• 负责人: Yohei Kirino
• 金额: $ 32.76万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158519
• 关键词: Address; Affect; Animals; Area; Biogenesis; Biological Assay; Biological Process; Bombyx; Bombyx mori; Cell Culture Techniques; Cells; Complex; Data; Defect; Development; Dissection; Drosophila genus; Embryo; Endoribonucleases; Ensure; Enzymes; Exonuclease; Female sterility; Generations; Genetic Transcription; Genome; Genomic Segment; Germ Cells; Goals; In Vitro; Knowledge; Length; Ligation; Malignant Neoplasms; Mediating; Methods; Methylation; Mitochondria; Mus; Nucleotides; Organism; Outer Mitochondrial Membrane; Ovary; Pathogenesis; Pathway interactions; Periodicity; Phenotype; Phosphorus; Process; Production; Proteins; RNA; RNA Binding; RNA Interference; RNA Precursors; RNA Sequences; Reaction; Research; Ribonucleases; Role; Sequence Analysis; Signal Pathway; Sterility; Testis; Transcript; Transfer RNA; Untranslated RNA; Up-Regulation; biological adaptation to stress; cancer type; endonuclease; experimental study; genetic information; genome integrity; hormonal signals; in vivo; inorganic phosphate; knock-down; knowledge base; next generation; novel; piRNA; reproductive system disorder; sex determination; stem; tool; transcriptome; transcriptome sequencing

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.

项目概要和摘要 以 Piwi（P 元素诱导的软睾）蛋白及其结合为中心的小调节 RNA 通路 Piwi 相互作用 RNA (piRNA) 沉默转座子和其他靶标，以维持动物基因组完整性 种系。 piRNA 通路的破坏导致转座子水平升高和种系缺陷 发育，最终导致动物不育。因此，piRNA 途径起到了守护者的作用。 种系基因组，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显而易见的，介导piRNA生物发生的因素和机制 仍然难以捉摸。 piRNA 的生物发生始于从定义的长单链 RNA 的转录 基因组区域称为 piRNA 簇。前体转录本可能被加工成更短的初级转录本 前体。然而，初级 piRNA 前体的加工概况和机制尚不清楚。 阐明了。初级前体由线粒体相关酶西葫芦进一步加工， 生成 pre-piRNA，将其加载到 Piwi 蛋白上，并在其中进行 3' 端加工并 甲基化成为成熟的 piRNA。我们的研究目标是阐明 piRNA 的生物发生途径， 我们一直在利用家蚕卵巢来源的 BmN4 细胞，该细胞内源性表达 Piwi 蛋白， piRNA。表达 piRNA 的可培养细胞的稀缺使得 BmN4 细胞适合并很好地用于 piRNA 生物起源研究。在细胞中，piRNA 由 tRNA 大量产生，我们最近揭示了 tRNA 衍生的 piRNA 的生物发生机制，其中 5'-tRNA 一半，而不是成熟的 tRNA，成为直接 piRNA 的前体。我们在 5'-tRNA 半部的 3' 末端发现了 2',3'-环状磷酸盐 (cP)，这促使我们 假设含有 cP 的 RNA (cP-RNA) 形成 piRNA 前体。我们的假设得到了加强 通过我们的进一步分析表明，在 BmN4 细胞中，cP-RNA 在外层大量积累。 piRNA 生物发生发生的线粒体膜，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 RNA-seq 无法捕获它们。我们开发了一种 cP-RNA-seq 方法，可以选择性地 测序cP-RNA，我们将利用它来全面鉴定Bombyx BmN4中表达的cP-RNA 细胞、果蝇 Kc167 细胞和小鼠睾丸，以研究它们与 piRNA（目标 1）。我们将进一步阐明 cP-RNA 与 Piwi 蛋白的相互作用以及 piRNA 生物发生 因子 BmPapi（目标 2），并表征 piRNA 生物合成中产生 cP-RNA 的 RNase（目标 3）。这 拟议的研究将推动我们的新研究工作，以了解 piRNA 生物发生的机制 专注于当前未表征的 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。