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MicroRNAs are endogenous regulatory non-coding RNA that exist in all multi-cellular organisms. Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however the in vivo functions of 3' non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3' non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3' non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets in vivo, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, we find that 3' non-seed pairing of let-7a can be functionally required even with sites that permit perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3' non-seed base-pairing can critically influence microRNA function in vivo.

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MicroRNAs (miRNA) 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 confer target repression. Recently, multiplede novocoding variants in the human Argonaute geneAGO1 (hAGO1)have been reported to cause a neurodevelopmental disorder (NDD) with intellectual disability (ID). Most of the altered amino acids are conserved between the miRNA-associated Argonautes inH. sapiensandC. elegans, suggesting thehAGO1mutations could disrupt evolutionarily conserved functions in the miRNA pathway. To investigate how thehAGO1mutations may affect miRNA biogenesis and/or functions, we genetically modeled four of thehAGO1 de novovariants (referred to as NDD mutations) by introducing the identical mutations to theC. elegans hAGO1homolog,alg-1. This array of mutations caused distinct effects onC. elegans miRNAfunctions, miRNA populations, and downstream gene expression, indicative of profound alterations in aspects of miRNA processing and miRISC formation and/or activity. Specifically, we found that thealg-1NDD mutations cause allele-specific disruptions in mature miRNA profiles both in terms of overall abundances and association with mutant ALG-1. We also observed allele-specific profiles of gene expression with altered translational efficiency and/or mRNA abundance. The sets of perturbed genes include human homologs whose dysfunction is known to cause NDD. We anticipate that these cross-clade genetic studies may advance the understanding of fundamental Argonaute functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.