Structural studies on macromolecular complexes in translational control and ribosome biogenesis

The ribosome is a large molecular machine central to the survival of all living cells. Genetic information is brought to the ribosome in the form of messenger RNA (mRNA) which is a mobile, molecular copy of a given gene sequence. The ribosome reads the information stored in the mRNA and translates it by synthesising a protein with a specific structure and function. By understanding the structure of a particular protein, we can often gain insights into its function (and malfunction in disease states) in the cell. Producing ribosomes requires a large proportion of a growing cell‘s resources and several genetic diseases are associated with inefficient ribosome production. Ribosome synthesis goes through a number of ordered steps, involving many proteins that ensure synthesis proceeds correctly. My research will examine two related aspects of protein production, firstly how a particular protein recognises certain mRNAs and prevents them from being translated. Interestingly, this protein is also hijacked by hepatitis C virus, which disguises its genes by mimicking cellular mRNA. The virus might use the normal function of this protein to aid its reproduction. Secondly, I will look at proteins involved in producing ribosomes and how they control the final stages of ribosome maturation.

Translation of messenger RNA (mRNA) into protein on the ribosome is a fundamental process at the core of cellular function. Ribosomes constitute a large protein-RNA assembly that, in eukaryotes, requires ~170 soluble factors during ribosome biogenesis. In eukaryotes, the export of pre-ribosomal particles across the nuclear envelope requires adaptor proteins to recruit components of the nuclear export machinery. Pre-ribosomal particles are only exported once they have reached a particular maturation state and translation does not occur until ribosome maturation is completed in the cytoplasm. The rate of translation of a given mRNA is dependent not only on its availability but also on protein factors bound to the mRNA that can either promote or inhibit translation, sequester mRNAs into stress granules or processing bodies, or otherwise alter the location of mRNAs within the cell. Many transiently expressed mRNAs contain cis-acting sequences in their 3‘ untranslated regions that mark them as unstable and recruit protein factors that activate mRNA turnover. Signalling events can induce binding of other protein factors that protect these mRNAs from degradation. The work proposed here will use a combination of biochemistry and X-ray crystallography to gain insights into the functions of macromolecular complexes involved in translational control of mRNAs and in ribosome biogenesis. Understanding the structures of proteins in different states and/or complexes often provides key insights into their function, and allows hypotheses about the molecular mechanisms of cellular processes to be developed. These hypotheses can then be tested by creating site-directed mutants that are examined in biochemical and cell-based assays. The first project examines how structured cis-acting sequences in certain mRNAs recruit a protein complex known as NF90/NF45. This complex promotes the stability of bound mRNAs and inhibits translation. This complex is also a cellular host factor recruited by hepatitis C virus to its RNA genome and co-localises at viral replication foci in infected hepatocytes. This project aims to understand the molecular basis for RNA recognition by NF90/NF45 in these different systems. The second project will address the molecular basis for the final maturation stages in eukaryotic ribosome biogenesis, based initially on studies in budding yeast. Removal and recycling of ribosomal export factors in the cytoplasm is often coupled to the insertion of ribosomal proteins. I will examine complexes of ribosomal processing factors with ribosomal proteins to understand the mechanism behind this functional coupling.