TRIM-NHL proteins are involved in a variety of functions from muscular and neuronal tissue development to controlling stem cell fate. While the NHL repeat domain (NRD) of these proteins was originally thought to be responsible for protein-protein interactions, several proteins in the TRIM-NHL family have been found to bind RNA with a specific motif through the NRD. The C. elegans protein NHL-2 is involved in both the miRNA and endogenous 22G RNA pathways, where it interacts with a DEAD-box RNA helicase (CGH-1 and DRH-3 respectfully). I have shown that like other TRIM-NHL proteins, NHL-2 binds to RNA with a U-rich motif, using the fluorescent anisotropy. Specific residues were identified as being key to NHL-2’s RNA binding capacity. These residues were then mutated and a significant reduction in NHL-2s RNA binding capacity was observed. The mutations will next be tested in vivo using C. elegans, where the phenotypes of RNA binding mutant worms can be compared to nhl-2 knock out to assess the biological importance of NHL-2s RNA binding.
Structural analysis of NHL-2 is also being undertaken, with aims to solve the structure of apo-NHL-2 and NHL-2 in complex with RNA. Currently the drosophila protein BRAT is the only TRIM-NHL protein to have the structure of its NRD solved. Solving the structure of NHL-2s NRD will provide insight into the differences between TRIM-NHL proteins that underlie their functional differences.