Inducing Mutations in the La Module of Human La-Related Protein 4 (HsLARP4) to Analyze its Interactions with Poly-A RNA

Human La-Related Protein 4 (HsLARP4) is a cytoplasmic, polyribosome-associated protein that forms part of a large family of proteins called La-related proteins. La-related proteins contain the well-conserved La-motif (LAM), which acts in synergy with the nearby RNA recognition motif (RRM) to bind RNA. The LAM and RRM, which together form the ‘La module’, are essential in the binding of HsLARP4 to poly-A RNA. While other La-related proteins interact with poly-U RNA, HsLARP4 only interacts with poly-A RNA. We hypothesize that inducing point mutations of several highly conserved residues on the La module of HsLARP4 (spanning residues 111-287) will affect the interaction of HsLARP4 with poly-A RNA. These conserved residues were identified through sequence alignment and the mutants were produced using custom-designed primers in the polymerase chain reaction (PCR). The mutants that were successfully cloned are: i) HsLARP4 (111-287) M160A, ii) HsLARP4 (111-287) Q126A, iii) HsLARP4 (111-287) D139A, iv) HsLARP4 (111-287) M160D, and v) HsLARP4 (111-287) D139I. After expressing and purifying the mutant proteins from E. coli, they were used in Isothermal Titration Calorimetry (ITC) to study the interactions between HsLARP4 and poly-A(15) RNA, since poly-A(15) RNA has been found to be the preferred RNA for optimum binding affinity with the wild-type HsLARP4 La module. The mutants were also analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy to assess the correct folding of the purified proteins. ITC experiments have been performed on three of the mutants, HsLARP4 (111-287) M160A, D139A, and Q126A, all of which showed no interactions with poly-A(15) RNA, suggesting that M160A, D139A, and Q126A are important residues for RNA recognition and binding. This work will increase our understanding of the family of La-related proteins and HsLARP4, and their interactions with poly-A RNA, all of which are significant factors in RNA stabilization and mRNA translation.