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

Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis 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 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 catalytic residues 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 contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

The catalytic activity of microRNA Argonautes plays a modest role in microRNA star strand destabilization in C. elegans.

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 genome editing in C. elegans to examine the role of miRNA-guided slicing in organismal development. In contrast to previous work, slicing-inactivating mutations 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 absence of slicing, 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 slicing for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on slicing. Gene expression changes were consistent with negligible to moderate loss of function for miRNA guides whose star strand was upregulated, suggesting a reduced proportion of mature miRISC in slicing mutants. 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, a factor in the Target-Directed miRNA Degradation (TDMD) pathway. Overall, this work defines a role for miRNA Argonaute slicing in star strand decay; future work should examine whether this role could have contributed to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.