The RNA-binding activity of the TRIM-NHL protein NHL-2 is essential for miRNA-mediated gene regulation

The conserved TRIM-NHL protein, NHL-2, plays a key role in small RNA pathways in Caenorhabditis elegans. NHL-2 has been shown to interact with U-rich RNA through its NHL domain, but the importance to its biological function is unknown. We defined the crystal structure of the NHL domain to 1.4 [A] resolution and identified residues that affect affinity for U-rich RNA. Functional analysis of an NHL-2 RNA-binding loss-of-function mutant demonstrated defects in the heterochronic pathway, suggesting that RNA binding is essential for its role in this miRNA pathway. Processing bodies were enlarged in the NHL-2 RNA-binding mutant, suggesting a defect in mRNA decay. We also identified the eIF4E binding protein IFET-1 as a strong synthetic interactor with NHL-2 and the DEAD box RNA helicase CGH-1 (DDX6), linking NHL-2 function to translation repression. We demonstrated that in the absence of NHL-2, there was an enrichment of miRNA transcripts associated with the miRNA pathway Argonaute proteins ALG-2 and ALG-2. We demonstrate that NHL-2 RNA-binding activity is essential for let-7 family miRNA-mediated translational repression. We conclude that the NHL-2, CGH-1, and IFET-1 regulatory axes work with the core miRISC components to form an effector complex that is required for some, but not all, miRNAs.