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

线虫种系发育过程中性二态性 piRNA 表达对内源基因的调节

• 批准号: 10389801
• 负责人: Margaret Starostik
• 金额: $ 4.68万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389801
• 关键词: Algorithms; Animals; Base Pairing; Binding; Biogenesis; Biological; Biological Assay; Biological Process; CRISPR/Cas technology; Caenorhabditis elegans; ChIP-seq; Complex; Consensus Sequence; DNA Transposable Elements; DNA-Binding Proteins; Data; Databases; Defect; Development; Disease; Drosophila genus; Elements; Embryonic Development; Epigenetic Process; Female; Fertility; Fertility Disorders; Gametogenesis; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Human; Individual; Infertility; Knowledge; Literature; Mammals; Mediating; Men' s Role; Modeling; Modification; Molecular; Mus; Mutagenesis; Mutate; Mutation; Nucleotides; Oogenesis; Pathway interactions; Pattern; Physiology; Process; Production; RNA; RNA Interference; Regulation; Regulatory Element; Reporter; Reporting; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Small Interfering RNA; Small RNA; Specific qualifier value; Sperm Maturation; Spermatogenesis; Testing; Therapeutic; Trans-Activators; Translations; Untranslated RNA; base; comparative; computerized tools; density; design; differential expression; experimental study; fly; genome integrity; human disease; improved; insight; mRNA Stability; mRNA sequencing; male; male fertility; mutant; novel; nuclease; preservation; promoter; selective expression; sex; sexual dimorphism; sperm cell; transcription factor; transcriptome sequencing

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.

项目摘要 小型RNA - 简短，非编码RNA - 是动物生理和疾病中关键的调节剂 通过完整的基础配对相互作用来控制mRNA稳定/翻译和表观遗传学的基因表达 修改。小型RNA介导的沉默途径在进化上是保守的，是最大的一类 小RNA包含PIWI相互作用RNA（PIRNA）。 Fly的广泛研究确定了Pirnas Silhouette 转座子并以性别特定的方式表达； PIRNA对于基因组完整性至关重要 种系开发。但是，哺乳动物和蠕虫中的大多数PIRNA都不映射到转座子，而是 预计将调节种系表达的基因。尽管单个PIRNA与 据报道，内源性基因，PIRNA的内源基因靶标在很大程度上未知。 在秀丽隐杆线虫中，约有15,000 piRNA中的每一个都是自主转录的，并包含上游顺式。 调节元件，红宝石主题。我们发现，在精子发生过程中，PIRNA的表达方式有所不同。 卵子发生和雄性PIRNA对红宝石基序中的5'C核苷酸具有很大的偏见。此外，我们 将SNPC-1.3鉴定为针对男性生育至关重要的性别特异性转录因子。 SNPC-1.3取决于已知的 核心PIRNA生物发生因子SNPC-4，可在精子发生过程中驱动雄性PIRNA的表达。损失SNPC- 1.3在精子发生过程中，雄性PIRNA和精子成熟缺陷的全球耗竭。其他 已经鉴定出了PIRNA生物发生因子，但是这些反式作用因子如何相互相互作用，并且 红宝石图案对此很了解。 在这项拟议的研究中，我假设性别特定的调节机制是pirna的基础 表达调节内源性基因对于适当的种系发育至关重要。测试我在 精子发生的背景，在目标1中，我将在目标和 使用核酸酶（切割和运行）释放以表征5 piRNA生物发生的蛋白质-DNA结合谱 Ruby Motif顺式调节元件的作用因子。此外，我将确定是否在 红宝石主题充当SNPC-1.3的男性特异性元素。我还将使用计算工具来识别推定的， 男性和雌性PIRNA的新型顺式调节元素。在AIM 2中，我将在计算上识别内源性 野生型和SNPC-1.3（ - ）动物中的小RNA-seq和mRNA-Seq的雄性PIRNA的基因靶标也 作为Pirtarbase，一个计算的数据库进行了预测和实验鉴定的PIRNA靶向位点。我 将使用记者测定法进行实验验证雄性PIRNA及其预测的内源基因靶标 内源性和合成PIRNA，并应用piRNA或其其crispr/cas9介导的诱变 目标。总的来说，这项研究将加强我们对性别特异性pirna表达方式的理解 在转录调节中，并提供了对piRNA的生物学作用的新见解，而不是转座子沉默。