Recognition of precursor microRNAs by the Dicer cofactor TRBP

MicroRNAs (miRNAs) are small non-coding RNAs that enable post-transcriptional gene regulation through RNA interference (RNAi). The biogenesis of miRNAs involves distinct enzymatic processing steps, each of which requires a specific set of proteins. The processing of precursor miRNAs (pre-miRNAs) is performed by the endoribonuclease Dicer. Dicer enzymes are conserved in all organisms that utilise RNAi and they always associate with a double-stranded (ds) RNA-binding protein. In mammals, Dicer associates with TAR element binding-protein (TRBP) but the precise role of TRBP in miRNA biogenesis remains unclear. Research over the last 15 years has suggested that TRBP aids Dicer substrate recognition, facilitates diffusion of Dicer along dsRNA substrates, influences the length of Dicer products, and regulates Dicer activity or stability. Human TRBP is a 366-residue protein that comprises three dsRNA-binding domains (dsRBDs) separated by long unstructured linkers. The two N-terminal Type-A dsRBDs bind dsRNA while the C-terminal Type-B domain mediates protein-protein interactions. The goal of this thesis is to explore the structure-function relationship and RNA-binding properties of TRBP. DsRBDs recognise dsRNA via three interaction surfaces: Regions 1-3. Previous research has highlighted the contribution of Region 2 and suggested that the sequence and molecular dynamics of this loop may tune the dsRNA-binding affinity of the domain. Here, nuclear magnetic resonance (NMR) spectroscopy was used to evaluate the structure and ps-ns backbone dynamics of the two Type-A dsRBDs of TRBP, with particular focus on RNA-binding region 2. Analysis of 15N NMR relaxation parameters and residue composition of this region revealed differences between the two dsRBDs. A series of single and double mutant variants were made in single and tandem dsRBD constructs, and their dsRNA-binding properties were tested using several pre-miRNAs. A previously reported difference in binding affinity between dsRBD-1 and 2 was shown to be dependent on pH, which was also shown to affect the foldedness of dsRBD-1. Single-site mutations were shown to alter the dynamics of Region 2 and to affect RNA-binding affinity. These analyses suggested that Region 2 may be required for sensing the presence of non-Watson-Crick features in a pre-miRNA. The implications of these results on Dicer activity are unclear, but the characterisation of binding properties and backbone dynamics reported here provide further insight into pre-miRNA recognition by TRBP and proteins with multiple dsRBD more generally.