Biogenesis of piRNA and its function in endogenous gene regulation

项目摘要 生殖系产生配子，将亲本的表观遗传和遗传信息传递给 后代生殖系的维持和功能性配子的发育需要Piwi， Argonaute家族的古老成员，以及Piwi相互作用小RNA（皮尔纳）。 与其他类型的小RNA（例如，microRNA和siRNA）、皮尔纳生物发生 仍然是神秘的，主要是由于它们在种系和序列中的排他性表达， 生物体之间的多样性。在过去的十年里，我们取得了重大进展， 阐明皮尔纳途径作为针对转座的防御机制的功能 元素然而，包括蠕虫和小鼠在内的生物体表达数千种piRNA， 对应于转座子序列，表明piRNA可能具有替代功能。 我的初步研究结果表明，C。线虫piRNA调控内源基因表达 并影响发育过程。该提案的目标是解决基本问题， 关于皮尔纳途径的问题：皮尔纳的表达是如何控制的， 转录水平？皮尔纳前体如何加工成成熟的piRNA？Do piRNAs 调节生殖细胞基因的表达它们是如何影响细胞和 发展过程？在K99阶段，我将：（1）定义 皮尔纳产生基因座的转录程序和与皮尔纳相关的染色质因子 （2）描述皮尔纳前体上的帽结构并研究其在皮尔纳中的作用 （3）建立无细胞系统以表征Piwi的酶活性， piRNA加工酶。在R 00阶段的奖励，我将：（1）继续在体外 生物化学研究;（2）确定piRNA如何调节发育过程， 剂量补偿;（3）研究Piwi表达的潜在自动调节。采取 总之，这项研究将为研究C. elegans线虫and in other species种类.这将极大地促进我们对机制的理解， piRNA在种系维持和配子发生中的功能。最终，这项工作将 提供新的工具和疗法，将改善人类生育能力和健康。

Project Summary The germline produces gametes that transmit parental epigenetic and genetic information to offspring. The maintenance of germline and the development of functional gametes require Piwi, an ancient member of the Argonaute family, and Piwi-interacting small RNAs (piRNA). Compared to other types of small RNAs (e.g., microRNAs and siRNAs), piRNA biogenesis remains mysterious largely owing to their exclusive expression in the germline and sequence diversity between organisms. Over the past decade, significant progress has been made toward elucidating the function of the piRNA pathway as a defense mechanism against transposable elements. Yet organisms including worms and mice express thousands of piRNAs that do not correspond to transposon sequences, suggesting that piRNAs may have alternative functions. My preliminary findings suggest that C. elegans piRNAs regulate endogenous gene expression and influence developmental processes. The goal of this proposal is to address fundamental questions regarding the piRNA pathway: How is piRNA expression controlled at the transcription level? How are piRNA precursors processed into mature piRNAs? Do piRNAs regulate the expression of germline genes? And how do they influence cellular and developmental processes? During the K99 phase of this award, I will: (1) define the transcription program of piRNA producing loci and chromatin factors associated with piRNA genes; (2) characterize a cap structure on piRNA precursors and study its role in piRNA expression; (3) established a cell free system to characterize enzymatic activity of Piwi and piRNA-processing enzymes. During the R00 phase of the award, I will: (1) continue the in vitro biochemical studies; (2) determine how piRNAs regulate developmental processes such as dosage compensation; (3) investigate potential auto-regulation of Piwi expression. Taken together, the proposed research will provide a framework for studying piRNA biogenesis in C. elegans and in other species. It will greatly advance our understanding of mechanism and functions of piRNAs in germline maintenance and gametogenesis. Ultimately the work will provide new tools and therapies that will improve human fertility and health.