Modeling neurodevelopmental disorder-associated human AGO1 mutations in C. elegans Argonaute alg-1. [ribosome profiling]. Modeling neurodevelopmental disorder-associated human AGO1 mutations in C. elegans Argonaute alg-1. [ribosome profiling]

MicroRNAs (miRNA) associate with Argonaute proteins and negatively regulate gene expression by base pairing with complementary sequences in the 3’ UTRs of target genes. De novo coding variants in the human Argonaute gene AGO1 were reported to cause neurodevelopmental disorder (NDD) with intellectual disability (ID). Most of the altered amino acids are conserved between the miRNA associated Argonautes in H. sapiens and C. elegans, suggesting that the human AGO1 mutations could disrupt evolutionarily conserved functions in miRNA biogenesis or target repression. We genetically modeled four human AGO1 mutations in C. elegans by introducing identical mutations into the C. elegans AGO1 homolog, ALG-1. These alg-1 NDD mutations caused phenotypes in C. elegans indicative of disrupted miRNA processing, miRISC formation, and/or target repression. We show that the alg-1 NDD mutations are antimorphic as they cause developmental and molecular phenotypes stronger than those exhibited by the alg-1 null mutants, likely by sequestrating functional miRNA silencing complex (miRISC) components into non-functional complexes that fail to confer robust gene repression. The alg-1 NDD mutations cause allele-specific disruptions in mature miRNA profiles, both in overall abundances and ALG-1 NDD association, accompanied by perturbation of downstream gene expression, including altered translational efficiency and/or mRNA abundance. The perturbed genes include those with human orthologs whose dysfunction is associated with NDD. These cross-clade genetic studies illuminate fundamental Argonaute functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms. Overall design: To profile the translatome and transcriptome of the alg-1 NDD mutants, we performed ribosome profiling (Ribo-seq) and RNAseq of the four alg-1 NDD mutants, the alg-1 null mutants, and WT C. elegans

miRNA（microRNA）可与Argonaute蛋白结合，并通过与靶基因3'非翻译区（3' untranslated region, 3' UTR）中的互补序列碱基配对，负向调控基因表达。据报道，人类Argonaute基因AGO1的从头编码变异可引发伴智力障碍（intellectual disability, ID）的神经发育障碍（neurodevelopmental disorder, NDD）。在人类（Homo sapiens, H. sapiens）与秀丽隐杆线虫（Caenorhabditis elegans, C. elegans）中，与miRNA结合的Argonaute蛋白的多数变异氨基酸位点均保守，这提示人类AGO1突变可能会破坏miRNA生物发生或靶基因抑制过程中进化保守的功能。我们通过在秀丽隐杆线虫的AGO1同源基因ALG-1中引入相同突变，在该物种中构建了4种人类AGO1突变的遗传模型。这些alg-1 NDD突变可在秀丽隐杆线虫中引发表型，提示miRNA加工、miRNA诱导沉默复合物（miRNA-induced silencing complex, miRISC）形成以及/或靶基因抑制过程受到破坏。我们证实，alg-1 NDD突变为显性负效突变：其引发的发育与分子表型较alg-1敲除突变体更为显著，其机制可能是通过将功能性miRNA沉默复合物（miRISC）组分隔离至无法有效介导基因抑制的非功能性复合物中。alg-1 NDD突变会引发成熟miRNA表达谱的等位基因特异性紊乱，包括整体丰度与ALG-1结合状态的改变，并伴随下游基因表达的扰动，具体表现为翻译效率和/或mRNA丰度的变化。这些受扰动的基因包含其功能异常与NDD相关的人类同源基因。这些跨进化枝的遗传学研究阐明了Argonaute蛋白的核心功能，并为理解miRNA介导的转录后调控机制的保守性提供了新视角。实验设计概述：为解析alg-1 NDD突变体的翻译组与转录组特征，我们对4种alg-1 NDD突变体、alg-1敲除突变体以及野生型（wild type, WT）秀丽隐杆线虫进行了核糖体谱分析（ribosome profiling, Ribo-seq）与RNA测序（RNA-seq）。