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 Argonautes是唯一可以使用任何RNA或DNA序列编程的蛋白质家族 制作转录，mRNA稳定性或翻译的序列特异性调节剂。我们的目标是了解 Argonaute蛋白质和途径的范式实例的生物学和机制，最终 使用这些见解来设计和改善针对人类疾病的小型RNA引导的疗法。确实，学习 Argonautes如何工作以及如何制作小型RNA指南导致了发展和FDA 批准小型RNA药物。然而，有关特异性和功能的基本问题 Argonaute蛋白介导的途径仍未得到解答。 例如，尽管进行了20年的研究，但我们仍然无法预测DICER酶将如何清除前 miRNA仅基于其序列。我们将使用生化和结构方法来识别特征 这确定了迪切尔在哪里切割前MIRNA以及迪切尔伴侣蛋白如何改变此过程。 在动物中，Argonaute蛋白的PIWI亚家族使用23–30-NT“ Pirna”指南沉默 转座子或调节生殖细胞中的基因表达。 PIRNA由特定的长，单链制成 前体RNA。我们的研究试图解释为什么某些基因组区域和成绩单注定为 制作PIRNA，而其他人则被排除在外。通过研究苍蝇，飞蛾和小鼠的胸肌，我们希望确定 动物用来指定Pirna前体的进化古老和新进化的策略， 将它们转化为具有PIWI蛋白的功能复合物。虽然实验和计算研究 已经显着提高了我们预测miRNA目标的能力，尚未取得类似的进步 pirnas。在胎盘哺乳动物的精子细胞中，Pachytene Pirnas几乎与核糖体一样丰富， 但是我们仍然不知道它们的规范。蛋白质中的突变使Pachytene piRNA 导致男性不育症，这表明Pachytene PiRNA促进了精子发育。我们将使用 生物化学和小鼠遗传学研究pachytene piRNA的功能和特异性。 最后，有30％的细菌基因组编码了Argonautes，但我们不知道它们做什么。出奇， 我们发现，在热嗜热中，DNA引导的，DNA旋转的Argonaute（Ttago）与 Gyrase A以确保成功复制。我们的假设是Ttago在解散该公司方面发挥了作用 DNA复制结束时的圆形染色体，也许是为了补偿没有 该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如何获得 它的指南，以及在体内如何和什么调节。 这些研究将共同​​揭示有机体用来制造小RNA的潜水策略以及如何 他们使用Argonautes来控制发展，差异化和复制健康。