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

项目摘要 Argonautes是唯一已知的可以用任何RNA或DNA序列编程的蛋白质家族， 使转录、mRNA稳定性或翻译的序列特异性调节器。我们的目标是了解 Argonaute蛋白质和途径的典型例子的生物学和机制，并最终， 利用这些见解来设计和改进针对人类疾病的小RNA指导疗法。事实上，学习 Argonautes的工作原理以及它们的小RNA向导是如何产生的， 小RNA药物的批准。尽管如此，关于特异性和功能的基本问题， Argonaute蛋白介导的途径仍然没有答案。 例如，尽管经过了20多年的研究，我们仍然无法预测Dicer酶如何切割一个前体， miRNA仅基于其序列。我们将使用生物化学和结构的方法来确定的特点， 它决定了Dicer切割pre-miRNA的位置以及Dicer伴侣蛋白如何改变这一过程。 在动物中，Argonaute蛋白质的PIWI亚家族使用23-30-nt“皮尔纳”指导沉默 转座子或调节生殖细胞中的基因表达。piRNA是由特定的长单链 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，而其他人则被排除在外。通过研究苍蝇、飞蛾和小鼠中的piRNA，我们希望找到 动物用来指定皮尔纳前体的进化上古老和新进化的策略， 将其转化为具有PIWI蛋白的功能复合物。虽然实验和计算研究 已经极大地提高了我们预测miRNA靶点的能力，类似的进展还没有取得， piRNA。在胎盘哺乳动物的精母细胞中，粗线期piRNA几乎与核糖体一样丰富， 但我们仍然不知道它们是如何调节的。制造粗线期piRNA的蛋白质中的突变 导致男性不育，表明粗线期piRNA促进精子发育。我们将使用 生物化学和小鼠遗传学来研究粗线期piRNA的功能和特异性。 最后，30%的细菌基因组编码Argonautes，但我们不知道它们做什么。令人惊讶的是， 我们发现，在嗜热栖热菌中，DNA引导的DNA切割Argonaute（TtAgo）与 促旋酶A以确保成功复制。我们的假设是，TtAgo在解开 在DNA复制结束时的环形染色体，也许是为了弥补缺乏的DNA。 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 它的指导，以及它在体内的调节方式和内容。 总之，这些研究将揭示生物体用于制造小RNA的不同策略，以及它们是如何制造小RNA的。 他们利用Argonautes来控制发育、分化和生殖健康。

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.