Regulation of endogenous genes by sexually dimorphic piRNA expression during germline development in C. elegans

项目摘要 小RNA--短的、非编码的RNA--在动物生理学和沉默疾病中是关键的调节者 互补碱基配对相互作用控制mRNA稳定性/翻译和表观遗传学的基因表达 修改。小RNA介导的沉默途径在进化上是保守的，并且是最大的一类 小RNA组成Piwi相互作用RNA(PiRNAs)。对果蝇的广泛研究证实，piRNAs沉默 转座子和以性别特异的方式表达；piRNAs对基因组完整性和 生殖系发育。然而，哺乳动物和蠕虫中的大多数piRNA并不映射到转座子，而是 预计将调控生殖系表达的基因。尽管单个piRNA与 内源基因已有报道，但piRNAs的内源基因靶点仍然很大程度上未知。 在线虫中，15,000个piRNA中的每个都是自主转录的，并包含一个上游顺- 监管元素，红宝石主题。我们发现，piRNAs在精子发生和发育过程中存在差异表达 卵子发生，雄性piRNAs对红宝石基序中的5‘C核苷酸有强烈的偏爱。此外，我们 鉴定SNPC-1.3是一种性别特异的转录因子，对男性生育能力至关重要。SNPC-1.3取决于已知的 核心piRNA生物发生因子SNPC-4，在精子发生过程中驱动男性piRNA表达。SNPC的损失- 1.3在精子发生过程中，会导致男性piRNAs的全面耗尽和精子成熟缺陷。其他 已经确定了pIRNA生物发生因子，但这些反式作用因子如何相互作用和 人们对红宝石的主题知之甚少。 在这项拟议的研究中，我假设pirna背后存在性别特异性的调控机制。 表达，以调节对生殖系正常发育至关重要的内源基因。来检验我的假设 在精子发生的背景下，在目标1中，我将使用最近开发的在靶和靶下切割的策略 用核酸酶释放法(Cut&Run)研究5piRNA生物发生反式病毒的蛋白质-DNA结合谱 红宝石基序顺式调控元件的作用因素。此外，我将确定5‘C核苷酸是否在 红宝石基序作为SNPC-1.3的男性特有元素。我还将使用计算工具来确定假定的、 男性和女性piRNA的新顺式调控元件。在目标2中，我将通过计算识别内源性 野生型和SNPC-1.3(-)动物的雄性小RNA-seq和mRNA-seq的基因靶点 作为piRTarBase，一个通过计算预测和实验确定的piRNA靶向位点的数据库。我 将使用报告分析对雄性piRNAs及其预测的内源性基因靶点进行实验验证 内源和合成的piRNAs及其在CRISPR/Cas9介导的piRNAs或其基因突变中的应用 目标。总而言之，这项研究将加强我们对性别特异性piRNA表达的理解 并提供了对转座子沉默以外的piRNAs生物学作用的新见解。

PROJECT ABSTRACT Small RNAs – short, noncoding RNAs – are critical regulators in animal physiology and disease that silence gene expression by complementary base pairing interactions to control mRNA stability/translation and epigenetic modifications. Small RNA-mediated silencing pathways are evolutionarily conserved, and the largest class of small RNAs comprise Piwi-interacting RNAs (piRNAs). Extensive studies in fly established that piRNAs silence transposons and are expressed in a sex-specific manner; piRNAs are essential for genome integrity and germline development. However, most piRNAs in mammals and worm do not map to transposons, and instead are predicted to regulate germline-expressed genes. Although novel associations of individual piRNAs with endogenous genes have been reported, endogenous gene targets of piRNAs remain largely unknown. In C. elegans, each of the ~15,000 piRNAs is autonomously transcribed and contains an upstream cis- regulatory element, the Ruby motif. We found piRNAs are differentially expressed during spermatogenesis and oogenesis, and male piRNAs have a strong bias for the 5’C nucleotide in the Ruby motif. Furthermore, we identified SNPC-1.3 as a sex-specific transcription factor critical for male fertility. SNPC-1.3 depends on a known core piRNA biogenesis factor, SNPC-4, to drive male piRNA expression during spermatogenesis. Loss of snpc- 1.3 during spermatogenesis results in global depletion of male piRNAs and sperm maturation defects. Other piRNA biogenesis factors have been identified, but how these trans-acting factors interact with each other and the Ruby motif is poorly understood. In this proposed research, I hypothesize that sex-specific regulatory mechanisms underlie piRNA expression to regulate endogenous genes critical for proper germline development. To test my hypothesis in the context of spermatogenesis, in Aim 1 I will use a recently developed strategy Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to characterize protein-DNA binding profiles of 5 piRNA biogenesis trans- acting factors at the Ruby motif cis-regulatory element. Furthermore, I will determine if the 5’ C nucleotide in the Ruby motif acts as a male specific element for SNPC-1.3. I will also use computational tools to identify putative, novel cis-regulatory elements for male and female piRNAs. In Aim 2, I will computationally identify endogenous gene targets of male piRNAs from small RNA-seq and mRNA-seq in wild-type and snpc-1.3(-) animals as well as piRTarBase, a database of computationally predicted and experimentally identified piRNA targeting sites. I will experimentally validate male piRNAs and their predicted endogenous gene targets using reporter assays for endogenous and synthetic piRNAs and applying CRISPR/Cas9-mediated mutagenesis of piRNAs or their targets. Collectively, this research will strengthen our understanding of how sex-specific piRNA expression is transcriptionally regulated and provide new insights into biological roles of piRNAs beyond transposon silencing.