Understanding the Evolution, Biology, and Molecular Mechanism of Argonaute Proteins

了解 Argonaute 蛋白质的进化、生物学和分子机制

• 批准号: 10210273
• 负责人: PHILLIP D ZAMORE
• 金额: $ 50.98万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-03 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210273
• 关键词: Animals; Bacteria; Bacterial Genome; Biochemical; Biochemistry; Biology; Chromosomes; Cleaved cell; Complex; DNA; DNA Sequence; DNA Topoisomerase IV; DNA biosynthesis; Development; Dicer Enzyme; Ensure; Evolution; Gene Expression; Genes; Genetic; Genetic Transcription; Genomic Segment; Germ Cells; Goals; Guide RNA; Male Infertility; Mediating; MicroRNAs; Molecular; Moths; Mus; Mutation; Organism; Pathway interactions; Pharmaceutical Preparations; Process; Protein Family; Proteins; RNA; RNA Precursors; RNA Sequences; Reproductive Health; Research; Ribosomes; Role; Small RNA; Specificity; Spermatocytes; Structure; Thermus thermophilus; Transcript; Translations; Work; base; computer studies; design; experimental study; fly; human disease; improved; in vivo; insight; mRNA Stability; mouse genetics; piRNA; placental mammal; plant fungi; pre-miRNA; sperm cell

Project Summary Argonautes are the only known family of proteins that can be programmed with any RNA or DNA sequence to make sequence-specific regulators of transcription, mRNA stability, or translation. Our goal is to understand the biology and mechanism of paradigmatic examples of Argonaute proteins and pathways, and, ultimately, to use these insights to design and improve small RNA-guided therapies for human diseases. Indeed, studying how Argonautes work and how their small RNA guides are made has led to the development and FDA approval of small RNA drugs. Nevertheless, fundamental questions about the specificity and function of Argonaute protein-mediated pathways remain unanswered. Despite >20 years of study, for example, we still cannot predict how Dicer enzymes will cleave a pre- miRNA based only on its sequence. We will use biochemical and structural approaches to identify the features that determine where Dicer cleaves a pre-miRNA and how Dicer partner proteins alter this process. In animals, the PIWI subfamily of Argonaute proteins uses 23–30-nt “piRNA” guides to silence transposons or regulate gene expression in germ cells. piRNAs are made from specific long, single-stranded precursor RNAs. Our research seeks to explain why some genomic regions and transcripts are destined to make piRNAs, while others are excluded. By studying piRNAs in flies, moths, and mice, we hope to identify both evolutionarily ancient and newly evolved strategies that animals use to designate piRNA precursors and to convert them into functional complexes with PIWI proteins. While experimental and computational studies have dramatically improved our ability to predict miRNA targets, similar advances have not yet been made for piRNAs. In the spermatocytes of placental mammals, pachytene piRNAs are nearly as abundant as ribosomes, but we still do not know what or how they regulate. Mutations in the proteins that make pachytene piRNAs cause male infertility, suggesting that pachytene piRNAs promote sperm development. We will use biochemistry and mouse genetics to study the function and specificity of pachytene piRNAs. Finally, 30% of bacterial genomes encode Argonautes, yet we do not know what they do. Surprisingly, we find that in Thermus thermophilus, the DNA-guided, DNA-cleaving Argonaute (TtAgo) acts together with gyrase A to ensure successful replication. Our hypothesis is that TtAgo has acquired a role in disentangling the circular chromosomes at the end of DNA replication, perhaps to compensate for the absence of Topoisomerase IV in this organism. We will use genetics and biochemistry to understand how TtAgo acquires its guides, and how and what it regulates in vivo. Together these studies will reveal diverse strategies that organisms use to make small RNAs and how they use Argonautes to control development, differentiation, and reproductive health.

项目摘要 ArgAertes是已知的唯一一个蛋白质家族，它可以用任何RNA或DNA序列编程来 对转录、信使核糖核酸的稳定性或翻译进行序列特异性调节。我们的目标是了解 ArgAerte蛋白质和途径的聚合例子的生物学和机制，并最终 利用这些洞察力来设计和改进针对人类疾病的小RNA引导疗法。的确，学习 阿戈宇航员是如何工作的，他们的小RNA指南是如何制作的，这导致了这种药物的开发和FDA 小RNA药物的批准。然而，关于生物多样性的特殊性和功能的基本问题 ArgAerte蛋白介导的途径仍未得到回答。 例如，尽管进行了20年的研究，但我们仍然无法预测迪格尔酶将如何裂解一种前 仅基于ITS序列的miRNA。我们将使用生化和结构方法来识别特征 这决定了迪特尔在哪里切割前miRNA，以及迪特尔伙伴蛋白如何改变这一过程。 在动物中，ArgAerte蛋白的PIWI亚家族使用23-30-nt的“piRNA”引导沉默 转座子或调节生殖细胞中的基因表达。PiRNA由特定的单链长链组成 前体RNA。我们的研究试图解释为什么一些基因组区域和转录本注定要 制造piRNA，而其他的被排除在外。通过研究苍蝇、飞蛾和老鼠的piRNA，我们希望能够识别 无论是进化上古老的还是新进化的策略，动物都使用这些策略来指定piRNA前体和 将它们转化为与PIWI蛋白的功能复合体。虽然实验和计算研究 极大地提高了我们预测miRNA靶标的能力，但类似的进展尚未取得 PIRNA。在胎盘哺乳动物的精母细胞中，粗线虫piRNAs几乎和核糖体一样丰富， 但我们仍然不知道他们监管什么或如何监管。制造粗线虫piRNA的蛋白质的突变 导致男性不育，这表明粗线虫piRNAs促进精子发育。我们将使用 生物化学和小鼠遗传学研究粗线虫piRNAs的功能和特异性。 最后，30%的细菌基因组编码Argavies，但我们不知道它们是做什么的。令人惊讶的是， 我们发现在Thermus thermophilus中，DNA引导的、DNA裂解的Argavite(TtAgo)与 旋转酶A，以确保复制成功。我们的假设是，TtAgo已经在解开 DNA复制末端的环形染色体，可能是为了弥补缺失 这种生物中的拓扑异构酶IV。我们将使用遗传学和生物化学来了解TtAgo是如何获得 它的指南，以及它在体内调节的方式和内容。 这些研究将一起揭示生物体用来制造小RNA的不同策略以及它们是如何 他们使用Argavies来控制发育、分化和生殖健康。