Dissection of the piRNA biogenesis pathway in germ cells

生殖细胞中 piRNA 生物发生途径的剖析

基本信息

批准号：
10402932
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10402932
关键词：
Address Affect Animals Area Biogenesis Biological Assay Biological Process Bombyx Bombyx mori 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

项目摘要

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.

项目摘要和摘要以Piwi（P元素诱导的Wimpy睾丸）蛋白及其结合的小型调节RNA通路为中心 PIWI相互作用的RNA（PIRNA）静音转座和其他靶标，以维持动物的基因组完整性种系。 PIRNA途径的破坏会导致转座子水平升高和种系缺陷开发，最终导致动物的不育。因此，Pirna途径是监护人生殖基因组以确保遗传信息传递到下一代。虽然至关重要 PIRNA在种系发育中的功能是介导PIRNA生物发生的因素和机制保持难以捉摸。 PIRNA的生物发生始于从定义的长单链RNA转录基因组区域称为piRNA簇。前体的成绩单可能被处理成较短的主要原则前体。但是，原代piRNA前体的处理的概况和机制不好阐明。通过线粒体相关的西葫芦酶进一步处理主要的前体生成Pre-PIRNA，这些PIRNA被加载到Piwi蛋白上，并在其中进行3'-End加工和甲基化成为成熟的PIRNA。朝着我们的研究目标阐明PIRNA的生物发生途径，我们一直在使用Bombyx Mori卵巢衍生的BMN4细胞，该细胞内源表达Piwi蛋白和 pirnas。表达piRNA的可培养细胞的稀缺性使BMN4细胞适合培养生物发生研究。在细胞中，PIRNA是由TRNA彻底产生的，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trnas成为直接 PIRNA的前体。我们发现5'-tRNA半末端的2'3'周期磷酸盐（CP）促使我们假设含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绝对积累在外部线粒体发生piRNA生物发生的膜，这些CP-RNA序列是与PIRNA序列重叠。 CP-RNA形成转录组的隐藏层，因为标准RNA-Seq无法捕获它们。我们已经开发了一种CP-RNA-seq方法，可以选择性地序列CP-RNA，我们将利用它来全面识别Bombyx BMN4中表达的CP-RNA 细胞，果蝇KC167细胞和小鼠测试，以研究其与 PIRNA（目标1）。我们将进一步阐明CP-RNA与PIWI蛋白和PIRNA生物发生的相互作用因子BMPAPI（AIM 2），并表征了PIRNA生物发生中产生CP-RNA的RNase（AIM 3）。拟议的研究将推动我们新的研究工作，以了解PIRNA生物发生的机理专注于当前纯净的CP-RNA，并支持生物医学目标以了解发病机理生殖系统疾病和癌症的癌症，这些疾病表达PIWI蛋白。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Exploration of CCA-added RNAs revealed the expression of mitochondrial non-coding RNAs regulated by CCA-adding enzyme.

对添加 CCA 的 RNA 的探索揭示了添加 CCA 的酶调节线粒体非编码 RNA 的表达。

DOI：
10.1080/15476286.2019.1664885
发表时间：
2019
期刊：
RNA biology
影响因子：
4.1
作者：
Pawar,Kamlesh;Shigematsu,Megumi;Loher,Phillipe;Honda,Shozo;Rigoutsos,Isidore;Kirino,Yohei
通讯作者：
Kirino,Yohei

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.