Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans [miRNA]

Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use C. elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role may contribute to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny. To test the role of miRISC-mediated slicing in invertebrates, we used CRISPR/Cas9-mediated genome editing to mutate the DEDH catalytic tetrad to AEDH in the major C. elegans microRNA Argonautes, ALG-1 and ALG-2 (alg-1(AEDH); alg-2(AEDH)). To determine the impact of mutating Argonautes’ catalytic residues on C. elegans microRNAs, we profiled small RNAs from embryos, L4 stage larvae and young adults. We also profiled small RNAs from embryos that were single mutants, with catalytic residues mutated in either ALG-1 or ALG-2. To test whether Target-directed microRNA degradation contributed to downregulation of select miRNAs in alg-1(AEDH); alg-2(AEDH), we added mutations to this background that theoretically inactivate all TDMD (ebax-1(null)) or TDMD of a mir-243 alone (Y47(mir-243bs_del)) and sequenced small RNAs in young adults.