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RNA--是动物生理学和疾病中的关键调节因子， 通过互补碱基配对相互作用控制mRNA稳定性/翻译和表观遗传的基因表达 修改.小RNA介导的沉默途径在进化上是保守的，并且是最大的一类沉默途径。 小RNA包括Piwi相互作用RNA（piRNA）。在果蝇中的广泛研究证实，piRNA沉默 转座子，并以性别特异性的方式表达; piRNA是基因组完整性所必需的， 生殖系发育然而，哺乳动物和蠕虫中的大多数piRNA不映射到转座子，而是 被预测调节生殖细胞表达的基因。虽然个别piRNA与 尽管已经报道了多种内源基因，但是piRNA的内源基因靶标仍然在很大程度上是未知的。 In C.在线虫中，约15，000个piRNA中的每一个都是自主转录的，并且含有上游顺式- 调控元件，红宝石基序。我们发现piRNA在精子发生过程中差异表达， 在卵子发生中，雄性piRNA对Ruby基序中的5 'C核苷酸具有强偏好。而且我们 将SNPC-1.3鉴定为对男性生育力至关重要的性别特异性转录因子。SNPC-1.3依赖于已知的 核心皮尔纳生物发生因子SNPC-4在精子发生期间驱动雄性皮尔纳表达。失去snpc- 1.3在精子发生过程中，导致雄性piRNA的全面耗尽和精子成熟缺陷。其他 已经鉴定了皮尔纳生物发生因子，但是这些反式作用因子如何彼此相互作用， 人们对红宝石图案了解甚少。 在这项拟议的研究中，我假设皮尔纳的性别特异性调控机制 表达以调节对于适当的种系发育至关重要的内源基因。为了验证我的假设 在精子发生的背景下，在目标1中，我将使用最近开发的策略靶下切割， 使用核酸酶（CUT&RUN）表征5种皮尔纳生物合成反式- 在Ruby基序顺式调节元件的作用因子。此外，我将确定是否5' C核苷酸在 红宝石基序作为SNPC-1.3的雄性特异性元件。我也会用计算工具来确定， 用于雄性和雌性piRNA的新型顺式调节元件。在目标2中，我将通过计算确定内源性 来自野生型和snpc-1.3（-）动物中的小RNA-seq和mRNA-seq的雄性piRNA的基因靶标 作为piRTarBase，计算预测和实验鉴定的皮尔纳靶向位点的数据库。我 将使用报告基因测定实验验证雄性piRNA及其预测的内源性基因靶标， 内源性和合成的piRNA以及应用CRISPR/Cas9介导的piRNA或其突变 目标的总的来说，这项研究将加强我们对性别特异性皮尔纳表达的理解， 本发明的目的是提供对转录调控的piRNA的新认识，并提供对转座子沉默之外的piRNA的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.