How do microRNAs regulate translation?

Recently, a completely new way of controlling gene expression has been identified. This has emerged as a result of the discovery of a whole new class of genes which, unlike most genes, do not produce proteins. Instead they make very small RNA molecules, called microRNAs. There are at least 700 microRNAs within the human genome, each of which has a different function. They work by binding to the messenger RNA molecules expressed from other genes and inhibiting the production of the proteins made from these genes. Each of these 700 small molecules is believed to interact with hundreds of other genes, thus adding to the complex regulation of the human genome. Already it has become clear that malfunction of microRNA regulation is associated with a growing list of human diseases, including cancer, diabetes, and viral infections. Importantly, the mechanisms by which these small RNA molecules inhibit the expression of other genes are not known. To develop new drugs it is essential for us to understand this process and how any malfunctions influence human disease. The experiments proposed here will allow us to discover what these mechanisms are.

MicroRNAs are non-coding, 21-25 nucleotide RNA molecules that base pair imperfectly to the 3' UTRs of target mRNAs and repress synthesis of the corresponding proteins. Approximately 700 miRNAs have been identified in humans and it has been estimated that they regulate at least 60% of all protein coding mRNAs. Already it has become clear that malfunction of microRNA regulation is associated with a growing list of human diseases, including cancer, diabetes and viral infection. Moreover, many major pharmaceutical companies are interested in developing microRNAs therapies to modulate gene expression and so treat a variety of diseases. However, for the development of specific pharmacological inhibitors that target these small RNAs it is necessary to characterise the mechanism(s) used by microRNA to regulate gene expression. Recently we have found that two distinct types of microRNA repression exist, with one acting at the initiation of protein synthesis and the other at a post-initiation event (Kong et al., 2008, PNAS). At present it is unclear how these two types of microRNA repression regulate translation and, indeed, many potential mechanisms have been proposed for each type of repression without a clear answer coming to light. The aim of this proposal is to systematically examine each step in the translation process, either to eliminate or define its involvement in the repression mechanism. The goals of this fellowship are broken down into three distinct sections to determine:

1) Which components of the cap-binding complex are regulated by miRNAs?
2) Does initiation play any functional role in the post-initiation repression?
3) Do members of the Argonaute (Ago) family of proteins play a direct role in translational regulation and in translational repression by microRNAs? In particular do these proteins regulate translation at the level of [GTP.eIF2.Met-tRNAi] ternary complex formation and do microRNAs affect this activity?