Investigations into RNA-binding proteins involved in eukaryotic gene regulation

Abstract

The flood of RNA-related research in recent decades has revealed RNA to be a structurally and functionally diverse class of molecule, one that generates an intricate network of regulation that has been pivotal to the evolution of complex lifeforms. In order to elucidate how RNA achieves biological function through the formation of ribonucleoprotein (RNP) complexes, characterisation of RNA recognition by RNA-binding proteins (RBPs) is an essential step. The rules governing the interaction of RNA and RBPs have proved difficult to define, and in many instances, it is not understood how specificity is achieved. Knowledge of these rules is crucial to our understanding of RNA-related functions and their role in disease, and requires further in-depth characterisation of a wide variety of RNP complexes. The research in this Thesis details the RNA-binding behaviour of two reported RBPs. Firstly, the RNA-binding behaviour of the Drosophila transcription factor bicoid is investigated. For many years it has been believed that the bicoid homeodomain binds the 3′-UTR of the caudal mRNA transcript, yet no binding site or specificity determinants have been reported. The work here attempts to characterise this interaction. Further, other domains in the protein are examined with a view to understanding how biological specificity might be achieved. Secondly, characterisation of the RNA-binding behaviour of the heterodimeric pair of transcription elongation factors, Spt4 and Spt5, is reported. This heterodimer is known to be an important player in transcription and yet remarkably little is known about its function. In the present work, the AA-repeat RNA-binding properties of these proteins are investigated, and complex binding behaviour is reported. Overall, it is shown that the elucidation of RNA-binding activity by proteins is often not straightforward, requiring the application of multiple and increasingly sophisticated techniques if we are to grasp the underlying biology