GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis

MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.微小RNA（MicroRNAs）与Argonaute蛋白（Argonaute）共同构成miRNA诱导沉默复合体（microRNA induced silencing complex, miRISC），该复合体可招募GW182蛋白（GW182），进而介导mRNA降解与/或翻译抑制。尽管该通路具有明确的保守性与重要的分子生物学意义，但在动物发育过程中，miRISC与GW182的相互作用是否为基因沉默所必需，目前仍未明确。本研究以秀丽隐杆线虫（Caenorhabditis elegans）为模型探究该问题，针对GW182的直系同源蛋白AIN-1与AIN-2，以及miRNA特异性Argonaute蛋白ALG-1之间的关联及其对基因沉默的影响展开分析。基于人类Argonaute蛋白结构的同源建模结果显示，ALG-1拥有保守的色氨酸结合口袋（Tryptophan-binding Pockets），该结构域是其与GW182结合所必需的。体外（in vitro）与体内（in vivo）实验证实，该结合口袋的突变会显著削弱ALG-1与AIN-1、AIN-2的结合能力。ALG-1色氨酸结合口袋突变体线虫仍保留miRNA结合与加工能力，但丧失了报告基因沉默活性。有趣的是，ALG-1色氨酸结合口袋突变体在幼虫阶段的表型与alg-1基因缺失突变体一致，却可挽救胚胎致死表型，这表明在该发育阶段，GW182同源蛋白与miRISC的结合并非必需。进一步研究发现，ALG-1色氨酸结合口袋突变体仍可调控胚胎期mir-35微小RNA家族的靶基因，这进一步证实了GW182蛋白在miRNA调控中的非必需性。综上，本研究结果表明，在秀丽隐杆线虫胚胎发育过程中，miRNA通路可独立于GW182蛋白发挥功能。