Modulation of the microRNA-induced silencing complex loading through a specific interaction with a J-domain co-chaperone

MicroRNAs (miRNAs) are essential regulators of several biological processes. To achieve their repressive function, they are loaded onto Argonaute (AGO) proteins, forming the microRNA Induced Silencing Complex (miRISC). Thus, regulating this loading step is essential for miRNA-dependent gene regulation. In this study, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans (C. elegans). Importantly, DNJ-12 does not interact with other AGOs, making it a specific regulator of miRNA function. Furthermore, the loss of DNJ-12 leads to developmental phenotypes previously shown to be dependent on miRNA function. Using the Auxin Inducible Degron (AID) system, a powerful tool to study the spatiotemporal effects of DNJ-12 depletion on miRNA function, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to a decrease in the levels of several miRNAs and prevents their loading onto ALG-1. This study highlights for the first time the requirement of a co-chaperone for the loading of miRNAs in vivo, provides a potential mechanism explaining AGO differential loading by small RNA, and therefore helps expand our understanding of the miRISC function in animals. We compared the effect of DNJ-12 depletion on microRNA expression in different C. elegans tissues

微小RNA（MicroRNAs, miRNAs）是多种生物学过程的关键调控因子。为发挥其基因沉默功能，miRNA会被装载至Argonaute（AGO）蛋白上，形成微小RNA诱导沉默复合体（microRNA Induced Silencing Complex, miRISC）。因此，调控该装载步骤对于依赖miRNA的基因调控至关重要。本研究鉴定出分子伴侣辅助因子DNJ-12作为ALG-1的新型互作蛋白，ALG-1是秀丽隐杆线虫（Caenorhabditis elegans, C. elegans）中两种主要的miRNA特异性AGO蛋白之一。值得注意的是，DNJ-12仅与ALG-1发生互作，而不结合其他AGO蛋白，使其成为miRNA功能的特异性调控因子。进一步研究发现，DNJ-12的缺失会导致此前被证实依赖miRNA功能的发育表型。本研究借助生长素诱导降解标签系统（Auxin Inducible Degron, AID）——这一用于研究DNJ-12缺失对miRNA功能时空影响的有力工具——证实DNJ-12缺失会阻碍ALG-1与热休克蛋白70（HSP70）的结合，而HSP70是体外实验中miRISC装载过程所需的分子伴侣。此外，DNJ-12缺失会导致多种miRNA的表达水平下降，并阻碍其被装载至ALG-1中。本研究首次揭示了分子伴侣辅助因子在体内miRNA装载过程中的必要性，阐明了小RNA介导AGO蛋白差异化装载的潜在机制，从而加深了我们对动物体内miRISC功能的理解。本研究还比较了DNJ-12缺失对秀丽隐杆线虫不同组织中miRNA表达的影响。