Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. Caenorhabditis elegans

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-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 population associated with ALG-1(anti) complexes in vivo. 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. Overall design: Deep-sequencing was performed on cDNA libraries made from total RNA and RNA immunoprecipitated with ALG-1 from mixed-staged populations of three strains: three biological replicates from wild-type animals and two biological replicates each from alg-1(ma192) and alg-1(ma202) mutant animals. In addition, deep-sequencing was performed on cDNA libraries made from L2-staged total RNA in two biological replicates from wildtype and alg-1(ma202) animals and one biological replicate of alg-1(ma192).

微小RNA（microRNAs，miRNAs）是基因表达的调控因子，其功能对于正常发育与生理稳态至关重要。我们此前已对秀丽隐杆线虫（Caenorhabditis elegans）中微小RNA特异性Argonaute蛋白（Argonaute）ALG-1（Argonaute-like gene）的反形态突变体（antimorphic）[alg-1(anti)]进行了功能鉴定。alg-1(anti)突变体所呈现的微小RNA相关表型，相较于完全缺失ALG-1的动物更为显著。ALG-1(anti)介导的微小RNA诱导沉默复合体（microRNA induced silencing complex，miRISC）无法完成从微小RNA加工到靶基因沉默的功能性转变。

为深入解析这一转变过程，我们对体内与ALG-1(anti)复合体结合的小RNA群体进行了功能鉴定。研究发现，alg-1(anti)突变体中微小RNA*（过客链，passenger strand）的积累量显著升高；免疫沉淀获取的ALG-1(anti)复合体中，成熟微小RNA与微小RNA*链的占比不符合化学计量比，部分微小RNA*链在ALG-1(anti) Argonaute蛋白中的含量远高于对应成熟微小RNA。

我们的研究揭示了微小RNA链选择与微小RNA*链处置过程中存在复合体特异性及微小RNA特异性的缺陷。对于部分微小RNA（如mir-58），ALG-1(anti)对微小RNA引导链的选择过程看似正常，但微小RNA*链的释放效率低下；而对于另一些微小RNA（如mir-2），ALG-1(anti)会同时选择微小RNA链与微小RNA*链作为引导链，这表明其链选择的正常特异性存在缺陷。

我们的研究结果表明，野生型ALG-1复合体在完成miRISC成熟过程中的链选择与微小RNA*链排出步骤时，可识别特定微小RNA的结构特征。

整体实验设计：我们从三种品系的混合阶段种群中提取总RNA，并获取经ALG-1免疫沉淀的RNA，分别构建cDNA文库并进行深度测序：其中野生型动物设置3个生物学重复，alg-1(ma192)与alg-1(ma202)突变体动物各设置2个生物学重复。此外，我们还从L2阶段的样本中提取总RNA，构建cDNA文库并进行深度测序：野生型与alg-1(ma202)动物各设置2个生物学重复，alg-1(ma192)设置1个生物学重复。