microRNAs (miRNAs) are crucial for normal development and physiology. To identify factors that might coordinate with miRNAs to regulate gene expression, we used 2O-methylated oligonucleotides to precipitate Caenorhabditis elegans let-7, miR-58, and miR-2 miRNAs and the associated proteins. A total of 211 proteins were identified through mass-spectrometry analysis of miRNA co-precipitates, which included previously identified interactors of key miRNA pathway components. Gene ontology analysis of the identified interactors revealed an enrichment for RNA binding proteins, suggesting that we captured proteins that may be involved in mRNA lifecycle. To determine which miRNA interactors are important for miRNA activity, we used RNAi to deplete putative miRNA co-factors in animals with compromised miRNA activity and looked for alterations of the miRNA mutant phenotypes. Depletion of 25 of 39 tested genes modified the miRNA mutant phenotypes in three sensitized backgrounds. Modulators of miRNA phenotypes ranged from RNA binding proteins RBD-1 and CEY-1 to metabolic factors such as DLST-1 and ECH-5, among others. The observed functional interactions suggest widespread coordination of these proteins with miRNAs to ultimately regulate gene expression. This study provides a foundation for future investigations aimed at deciphering the molecular mechanisms of miRNA-mediated gene regulation.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.