RNA helicases are the largest group of RNA interacting proteins in eukaryotes and are a vital mediator to the mRNA pathway. A key mediatory step in the mRNA pathway is the swift removal of mRNA possessing a premature termination codon (PTC). This is performed to prevent the formation of C-terminus truncated proteins which may have detrimental effects on the cell. The breakdown of PTC containing mRNA is performed by the nonsense mediated decay (NMD) pathway. Failures in this pathway can lead to many developmental issues and life-long conditions. One of the RNA helicases involved in this pathway is the DEAH-box 34 RNA helicase (DHX34). DHX34 has been shown to be key to the recruitment of SMG1 to UPF1, two key proteins in the NMD complex formation at the core of the PTC containing mRNA breakdown, and the subsequent phosphorylation of UPF1 to induce this mRNA decay. Although its role in the NMD pathway has been shown, and conditions have been linked to mutation in DHX34, its molecular mechanism and RNA remodelling activity are unknown. In this thesis, I use the optical tweezers single-molecule force-spectroscopy approach to investigate the interaction of DHX34 with RNA. I begin by designing and testing a novel assay to function within the optical tweezers and experimental requirements of this thesis. Next, I investigate DHX34’s ability to remodel duplex RNA through processive remodelling utilising a force-clamp assay. Here I demonstrate, through a lack of unwinding, that DHX34 is unlikely to be processive, but observe the remodelling of the hairpin beyond the immediate site of tension. Finally, I redesign the experimental approach to assess the remodelling activity of non-processive helicases using optical tweezers, achieved by introducing force-driven unfolding steps with the protein presence. This redesign, and the collected results, suggests that DHX34 preferentially binds ssRNA and most likely
5
maintains this through ATP turnover, and indicates an intermediate state to the currently recognised RNA helicase molecular mechanism pathway.