Development of Single-Molecule Methods for RNA Splicing

RNA splicing is an important step in the synthesis of most mammalian proteins and understanding the underlying molecular mechanisms is critical for tackling diseases linked to splicing. An important determinant is SRSF1, which has multiple roles in constitutive and alternative splicing. One of these roles is in the recognition and selection of 5’ splice sites due to an interaction with U1 snRNP during formation of complexes E and A. The exact roles and modes of interaction are not clear. Single-molecule methods are a key to understanding these. In this work, single-molecule methods were developed to investigate the number of bound proteins under different conditions. A home-built microscope utilising objective-based illumination by total internal reflection was used to look at these interactions at a single-molecule level and investigate the number of bound proteins under different conditions. The results showed that there was a distinctive reduction in the number of bound proteins in complex A, dependent on the availability of ATP. This was linked to the number of functional 5’ splice sites present, the U1 snRNP and phosphorylation. We could not find any evidence that sequences known to mediate stimulation by SRSF1 affect its binding. Using total internal reflection has inherent limitations, among them the necessary surface attachment and the dilutions required. These limitations could be overcome by the use of isolated microenvironments in the form of tiny droplets. A robust and convenient microfluidic device with a feature size of 3μm was set up and a suitable surfactant for biological samples was identified. Droplets with a diameter of 1μm were generated for the first time using flow focussing and single quantum dots and fluorescent proteins where identified within these droplets. The fluorescence intensity time traces from these droplets enabled the number of encapsulated fluorescent particles to be measured.