Dynamics and mechanisms of Argonaute proteins and the RISC complex studied with single molecule fluorescence spectroscopy

来自所有三个生命领域的ArgAerte(AGO)蛋白在特定调节细胞核酸水平的过程中发挥着关键作用。其中一些AGO蛋白，包括人类Argavite2(HAGO2)和来自古生物甲烷杆菌Jannaschii(MjAgo)的AGO，能够切割通过AGO相关的短互补引导链识别的核酸靶链。人类AGO2和MjAGO是真核和原核AGO分支的代表，具有保守的四结构域结构。然而，eAGOS和PAGOS的生物学作用明显不同。作为真核RNA干扰途径的关键组成部分，hAGO2参与了高达60%的基因的转录后精细调节。这一过程中的失败可能会产生毁灭性的后果，从糖尿病、几种形式的癌症到心力衰竭。相比之下，原核生物AGO蛋白被认为参与了针对针对DNA而不是RNA的外来核酸的细胞防御，并能够以依赖于向导和不依赖于向导的方式进行操作。然而，对这一过程及其监管的了解仍然很少。我们发展了生化、结构和尖端光学单分子方法，使我们能够阐明原核生物和真核生物AGO蛋白的结构-功能-动力学关系。通过我们的研究，我们打算加深对eAGO和PAGO机制的理解，以及AGO蛋白在整个活性周期中采样的构象空间，从而揭示这些结构保守的蛋白质如何能够在生物高度多样化的途径中发挥作用。本实验室对MjAGO的晶体结构和功能研究表明，MjAGO具有一个被部分底物占据的二级核酸结合通道。我们将描述结合通道的占有率和伴随的构象变化。此外，我们的目的是阐明复杂底物(如质粒DNA)是如何在原核生物AGO中调节的，以及哪些调节因子控制MjAGO的作用。HAGO2的活性受多种蛋白质和核酸相互作用伙伴的调控，并受到翻译后修饰的调节。这些相互作用和修饰如何影响hAGO2的结构组织尚不清楚。我们的目标是捕捉含hAGO2的复合体的不同阶段，以使用单分子FRET测量来确定这些复合体的结构组织和动力学。另一个主要焦点将是在单分子水平上分析人类AGO蛋白与前处理核酸酶DICER之间的相互作用。

Argonaute (AGO) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these AGO proteins, among them human Argonaute2 (hAGO2) and AGO from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an AGO-associated short complementary guide strand. Human AGO2 and MjAGO are representatives of the eukaryotic and prokaryotic AGO clade that share a conserved four-domain architecture. However, the biological roles of eAGOs and pAGOS differ significantly. As key components of the eukaryotic RNA interference pathway, hAGO2 takes part in the finely tuned post-transcriptional regulation of up to 60% of our genes. Failure in this process can have devastating consequences ranging from diabetes and several forms of cancer to heart failure. In contrast, prokaryotic AGO proteins are implicated to partake in the cellular defence against foreign nucleic acids targeting DNA rather than RNA and are able to operate in a guide-dependent and guide-independent manner. The understanding of this process and its regulation, however, remains sparse. We have developed biochemical, structural and cutting-edge optical single-molecule methods that allow us to illuminate the structure-function-dynamics relationship of prokaryotic and eukaryotic AGO proteins. With our investigations we intend to deepen the understanding of eAGO and pAGO mechanisms and the conformational space sampled by AGO proteins throughout their activity cycle thereby shedding light on the question how these structurally conserved proteins are able to function in biologically highly diverse pathways. We will focus on the following questions:Crystal structures and functional studies of MjAGO from our laboratory indicate that MjAGO possesses a secondary nucleic acid binding channel that is occupied by a subset of substrates. We will characterise the binding channel occupancy and accompanying conformational changes. Furthermore, we aim to elucidate how complex substrates (e.g. plasmid DNA) are accommodated in prokaryotic AGOs and which regulating factors control the action of MjAGO. The activity of hAGO2 is orchestrated by various protein and nucleic acid interaction partners and modulated by post-translational modifications. How these interactors and modifications influence the structural organisation of hAGO2 is poorly understood. We aim to capture different stages of hAGO2-containing complexes to determine the structural organisation and dynamics of these complexes employing single-molecule FRET measurements. An additional major focus will be the analysis of the interaction between human AGO proteins with the pre-processor nuclease Dicer on the single-molecule level.