Abstract
Human La-related protein 6 (HsLARP6) regulates the highly organized biosynthesis of type I procollagen polypeptides and affects the proper assembly of procollagen peptides into heterotrimers of type I procollagen. HsLARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop (5'SL) motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic propensity of the La domain of HsLARP6 to aggregate hampers studies probing the molecular basis of biologically and disease-relevant structure-function relationships, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effects of the protein construct length and RNA binding on protein stability. C-terminal truncations greatly impact protein stability, while N-terminal truncations have little to no effect on protein aggregation and RNA binding. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50 times longer than the unbound state under identical conditions. These results provide a significant platform for further studies of the molecular recognition of 5'SL by HsLARP6.