Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal.
Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal.
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秀丽隐杆线虫 ALG-1 反态突变揭示了 Argonaute 在 microRNA 引导链选择和过客链处理中的功能。
DOI：
10.1073/pnas.1506576112
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发表时间：
2015
影响因子：
11.1
通讯作者：
Ambros,VictorR
中科院分区：
文献类型：
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作者：
Zinovyeva,AnnaY;Veksler-Lublinsky,Isana;Vashisht,AjayA;Wohlschlegel,JamesA;Ambros,VictorR
MicroRNA 是基因表达的调节因子，其功能对于正常发育和生理至关重要。我们之前已经描述了秀丽隐杆线虫microRNA特异性Argonaute ALG-1（Argonaute样基因）中的突变，这些突变是反态[alg-1（抗）]的。alg-1（抗）突变体比完全丧失ALG-1的动物具有更强的microRNA相关表型。 ALG-1（抗）miRISC（microRNA 诱导沉默复合物）未能经历从 microRNA 处理到目标抑制的功能转变。为了更好地理解这种转变，我们对体内与 ALG-1(抗) 复合物相关的小 RNA 和蛋白质群体进行了表征。我们广泛表征了与野生型和突变型 ALG-1 相关的蛋白质，发现突变型 ALG-1(抗) 蛋白无法与许多 miRISC 辅因子相互作用，包括已知的靶点抑制所必需的蛋白质。此外，alg-1（抗）突变体显着过度积累 microRNA*（过客）链，免疫沉淀的 ALG-1（抗）复合物含有非化学计量产量的成熟 microRNA 和 microRNA* 链，ALG-1（抗）Argonaute 中存在的一些 microRNA* 链远远超过相应的成熟 microRNA。我们展示了 microRNA 链选择和 microRNA* 链处置中复杂的和 microRNA 特异性的缺陷。对于某些 microRNA（例如 mir-58），ALG-1（抗）的 microRNA 引导链选择似乎正常，但 microRNA* 链释放效率低下。对于其他 microRNA（例如 mir-2），microRNA 和 microRNA* 链均被 ALG-1（抗）选择为指导，表明链选择的正常特异性存在缺陷。我们的结果表明，野生型 ALG-1 复合物在执行 miRISC 成熟的链选择和 microRNA* 喷射步骤的背景下识别特定 microRNA 的结构特征。
MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in aCaenorhabditis elegansmicroRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)].alg-1(anti)mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition,alg-1(anti)mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation.