Optimisation of heteromeric protein complex formation via co-ordinated mRNA localisation

The goal of the project is to understand how to optimally generate heteromeric protein complexes in yeast making use of cytosolic translation factories and the test case eIF3. Information gained will be applied to the production of heterologous heteromeric complexes that can be commercially important in yeast (e.g. haemoglobin and human gonadotropin). More widely though the lessons learned will be generally applicable to the production of heterologous proteins using yeast as a host organism. The biotechnology focus of this studentship makes it a good fit to the BBSRC high-level theme 2 "Tackling strategic challenges"

More specificially, in this project a student will study the co-ordinated localisation and translation of mRNAs to translation factories. The mRNAs to be studied encode subunits of heteromeric protein complexes, and the key question to be addressed is whether the folding, assembly and biological function of the resulting protein complexes are regulated as a consequence of production at a defined site in a cytosolic translation factory.

The eIF3, eIF2 and NatA complexes in yeast will be used as exemplar protein complexes where the mRNAs encoding the various subunits are localised to translation factories. eIF3 and eIF2 represent key complexes involved in the synthesis of proteins in the recruitment of the ribosome to the mRNA to be translated. NatA is an N-acetyl transferase complex responsible for the modification of proteins while they are being produced by the ribosome. All three complexes are highly abundant and important, so it is unsurprising that they form co-translationally at specific sites, but what is less clear is how such an arrangement is set up, organised and controlled. These studies will establish a set of rules that can be then applied to the production of biotechnologically relevant heterologous protein complexes - assessing whether targeting heterologous mRNAs to protein factories increases productivity and yield of protein complexes.

A student will be trained in a plethora of different methods and assays as part of this project. Live cell MS2-based imaging techniques and single molecule fluorescent in situ hybridisation will be used to establish the location of mRNAs in cells. Innovative fluorescent assays will be used to study the site of translation of each mRNA, and polysome analysis will be used to characterise the efficiency of translation. Biochemical assays will be used to study the proportion of each subunit that is present in the soluble or protein aggregate fractions, and in vitro assays using yeast translation extracts will be used to dissect the eIF3 assembly process.

The questions that will be addressed are:
1. What RNA elements and protein factors control the localisation and translation of the eIF3, eIF2 and NatA mRNAs?
2. Is eIF3, eIF2 and NatA production affected by conditions and mutations that alter the localisation of the mRNAs?
3. Does localisation to a translation factory alter the proportion of protein complex subunits that are present as soluble active forms versus insoluble aggregates especially under stress conditions?
4. Can the information gleaned for eIF3, eIF2 and NatA complex formation be used to direct the formation of translation factories for heterologous protein expression?