MicroRNAs are endogenous non-coding RNAs important for post-transcriptional regulation of gene expression. miRNAs associate with Argonaute proteins to bind to the 3’ UTR of target genes, and post-transcriptionally regulate gene expression. It is well established that base pairing between the mRNA target and the miRNA seed region (g2-g8) is essential for targeting, but the function of the 3’ non-seed region (g9-g22) of microRNAs is less well understood. In this thesis, we systematically investigated the biological functions of 3’ non-seed nucleotides of C. elegans let-7a as an example of an evolutionarily conserved miRNA. We show that the 3’ non-seed sequence of let-7a determines target specificity among its family paralogs, and that the identity of each nucleotide at g11-g16 is essential for in vivo function. We confirmed that lin-41, as well as other heterochronic genes, are let-7 targets that require the 3’ pairing, in many cases to g11-g16 nucleotides. To investigate how 3’ pairing functionally interacts with seed pairing, we systematically re-configured the let-7a::lin-41 interactions and found that the 3’ pairing is critically required for full repression efficacy in the context of perfect seed pairing as well imperfect seed pairing. Certain de novo mutations on human Argonaute AGO1 have been reported to cause neuronal developmental defects with intellectual disability (ID). Many of these human ID mutations alter amino acids that are conserved between human AGO1 and its C. elegans homolog, ALG-1. We found that these mutations can be hypomorphic and antimorphic when modeled in C. elegans ALG-1. The human AGO1 mutations modeled in C. elegans ALG-1 can impact miRNA biogenesis, and lead to global perturbance in the translatome and transcriptome. We also show that the mutant ALG-1 can bind to microRNAs in C. elegans, and in so doing sequester microRNAs in nonfunctional miRISC, causing phenotypes stronger than alg-1(null). We show that the different ID mutations modeled in ALG-1 can exhibit distinct molecular phenotypes in terms of specific disruptions of miRNA biogenesis, and gene expression, suggesting that the mutated residues critically contribute to particular in vivo functions of Argonaute. Notably, different ID mutations affect distinct ALG-1 functions to varying degrees. These studies may provide insights into miRNA-mediated gene-regulatory mechanisms and advance the understanding of fundamental Argonaute functions.