Identification and analysis of factors that regulate the activity of the yeast exosome complex of exoribonucleases

Understanding biological processes at the molecular level remains one of the greatest challenges of the modern age. In addition to understanding what makes us tick, fundamental biological research can potentially aid the treatment or prevention of human diseases, lead to increased agricultural production/diversity, support environment management and trigger the development of new biology-based technologies. Central to all cellular biological processes is the flow of genetic information through what is known as the gene expression pathway. In this pathway, the linear DNA sequence of our genes is copied into a chemically similar but less stable molecule, called RNA. These short-lived gene copies, known as messenger RNA (mRNA), are then transported to highly specialised structures called ribosomes. Here, the information encoded within the mRNA is converted into functional protein molecules, which then carry out the specific processes required in the cell. A mistake at any point within this pathway can have very serious consequences for the cell. For example, a copying error inserted into the mRNA sequence can result in an incorrectly folded or shortened protein that is unable to perform its function correctly. Mistakes in gene expression occur rather often, even in normal, healthy cells. Therefore, it is essential that cells are able to recognise faulty mRNA molecules and destroy them efficiently. To monitor the production of their mRNA, cells have evolved a number of quality control systems that are known collectively as mRNA surveillance. These processes ensure that incorrectly produced mRNA molecules are selected out and rapidly degraded, thereby preventing the production of defective proteins. A major piece of the cell's armoury for degrading RNA is the exosome. The exosome consists of 10 different enzymes associated with one another in a complex. Packaged together, these enzymes can work more efficiently than they could on their own. The complex also allows coordinated regulation; all the enzymes can be switched on or swiched off at the same time. Previous studies have identified the components of the exosome complex and demonstrated its function in RNA degradation. However, little is known about how the complex is regulated. This research project will identify proteins that function either to promote the activity of the exosome or to inhibit its activity. The function of the identified proteins in exosome-dependent cellular processes such as mRNA surveillance will then be studied. Further studies on mRNA surveillance will increase our understanding of how gene expression is controlled in the cell. This knowledge will be of potential benefit in the design and development of new biotechnological strategies for protein production. Moreover, mRNA surveillance phenomena are directly linked to the basis of some prevalent human genetic disorders, such as breast cancer. The exosome is also a biological target of a successful anti-cancer drug. Hence, a knowledge of how the activity of the exosome complex is regulated may provide an important resource of information in developing future strategies in the treatment or prevention of disease.

The exosome is a multienzyme complex of 3prime -5prime exoribonucleases that has been conserved throughout eukaryotic evolution. The exosome complex plays a key role in the 3prime end maturation of diverse stable RNAs, including 5.8S ribosomal RNA (rRNA), the small nuclear RNAs (snRNAs) required for pre-mRNA splicing and the small nucleolar RNAs (snoRNAs) that function in ribosome biogenesis. The exosome also functions in mRNA turnover pathways and plays a central role in RNA quality control pathways that degrade improperly processed or assembled RNAs. The diverse nature of the different exosome substrates and their very distinct fates (productive RNA synthesis or complete RNA degradation) suggests that the activity of the complex must be tightly regulated. How the activity of the exosome is regulated is, as yet, largely unknown. In vitro enzymatic studies on purified exosomes suggest two distinct regulatory mechanisms for this complex. Exosome complexes purified from cell lysates exhibit a rather weak exonucleolytic activity in vitro, given the robust nature of exosome-mediated processes in vivo. Biochemical fractionation studies have shown that a small subpopulation of exosome complexes exhibits very high specific activity. Since exosome complexes are reported to interact functionally with a number of different proteins, the high specific activity of this fraction of exosome complexes is predicted to be due to the presence of one or more additional, as yet uncharacterised, protein factors. The activity of exosome complexes can also be downregulated. Exosome complexes can bind to GTP and are specifically inhibited in the presence of this NTP. The research outlined in this proposal will employ nanoelectrospray MALDI/MS mass spectrometry and an established, robust in vitro exonuclease assay to identify proteins that copurify with the exosome complex and regulate its activity. Subsequent analyses on the identified proteins will characterise their biochemical function in vitro and analyse their role in exosome-mediated processes in vivo. It is estimated that proteins present in as little as approximately 1 percent of all exosome complexes will be readily detected by MALDI/MS in this study. Exosome complexes will be purified by affinity chromatography, using strains expressing an epitope-tagged form of the exosome component Rrp4p that contains a TEV protease recognition site. In vitro exonuclease assays will be performed on the affinity purified complexes using short, synthetic 5prime labelled RNA substrates and the reaction products will be resolved by PAGE. Exosome complexes with increased specific activity will be resolved by a combination of ion exchange chromatography and activity profiling. Exosome complexes and associated proteins will be resolved by PAGE and candidate regulatory proteins will be identified by MALDI/MS. Covalent exosome-GTP complexes will be generated by irradiation with ultraviolet light. Crosslinked proteins will be resolved from the exosome components by 2D-PAGE and identified by MALDI/MS, as above. Interactions between the exosome complex and the identified proteins will be confirmed by reverse tagging experiments, using specific antiserum raised against Rrp4p. The functions of the identified proteins will be analysed both in vitro and in vivo. The proteins will be expressed in recombinant form and tested for their putative function in vitro (stimulation of exosome exonuclease activity, GTPase activity or GTP binding). To analyse the protein's role in exosome function in vivo, RNA will be isolated from mutant strains before and after its genetic depletion or overexpression and analysed by Northern blot hybridisation. To analyse effects on mRNA surveillance pathways, experiments will be performed using available reporter constructs expressing defective mRNA transcripts or in characterised mutants that show conditional defects in mRNA synthesis or assembly.