Quantitative dissection of protein synthesis initiation at 'omic and single mRNA scales

Cells in all forms of life rely on proteins to provide structural components as well as to perform almost every varied function. In people this ranges from fundamental processes such as digesting food and extracting oxygen from the air to less well understood processes such as enabling our brains to both learn and recall information. Proteins are the functional units encoded in our genes and rely on complex machines called ribosomes to ensure that each gene sequence is accurately decoded from mRNA instructions every time. Ribosomes are guided to the correct places on mRNAs by dedicated protein synthesis factors, themselves made of proteins, as well as other proteins that act by interacting with ribosomes and/or mRNAs. Many of the players act in ways that are not fully understood. Together they orchestrate a complex series of processes which ensure that every cell has the proteins needed at the right time and place to perform an array of functions. When processes are defective this can lead to disease. Because protein synthesis underpins all cellular activity it is critical that we understand precisely the rules that govern the mechanism and how it is controlled. A generic pathway describing the protein synthesis process has been uncovered. However, this stems mainly from studying the synthesis of a small number of proteins. Recent evidence from our labs and from others suggests this is overly simplistic and there may be several different ways to promote the recruitment of ribosomes to mRNAs in cells.

In this proposal we describe an approach aimed to quantify the amount of each protein and RNA present in different complexes and intermediates formed during the first stage of protein synthesis: initiation. Our approach combines for the first time the use of modern mass spectrometry techniques using known amounts of mass standards to quantify absolute amounts of each protein component with RNA sequencing to quantify the RNAs within a range of separable translation complexes isolated from cells. In addition, we will use newly developed microscopy approaches using fluorescent labels to visualise interactions between protein synthesis factors and individual mRNAs in growing cells. Our approaches therefore combine complimentary global approaches with directed single mRNA studies. These experiments will be done using actively translating cells as well as those undergoing cell stresses that rapidly reprogram protein synthesis and so change the interactions studied.

Together the proposed work will address important outstanding questions including whether the first engagement of ribosomes with newly made mRNAs uses the same factors and stoichiometry as subsequent initiation events on the same mRNA. Whether cell stresses that inhibit bulk protein synthesis but favour selective ribosome engagement do so with altered stoichiometry and mechanisms. Our single RNA approaches will provide mechanistic example mRNAs that follow distinct pathways to being synthesized. As a whole the program will provide mechanistic insights into the 'rules of life'. As protein synthesis defects are implicated in an increasing number of human disorders the insights gained may be broadly of value in understanding their mechanisms. In addition, many biotechnology applications rely on purified proteins and insight gleaned from this application into mechanisms may assist in the design of expression systems. Finally, the methodological approaches we implement will be more widely applicable to other cellular processes.

Quantitative biology relies on accurate descriptions of biological processes at multiple levels including understanding of the macro-molecular interactions between proteins and RNAs that govern key cellular processes such as protein synthesis. Here we aim to define the stoichiometry and absolute protein composition of protein and protein-RNA complexes, thereby the rules that underpin the mechanisms of translation initiation during optimal growth and during stress conditions that reprogram gene regulation at the level of protein synthesis to aid cell recovery and survival. By studying acute stresses with distinct translation factor targets we will gain insights into factors important for cellular reprogramming events that ensure specific mRNAs remain translationally active when others are repressed. The results obtained will mechanistically distinguish between several current competing models of the pathway for protein synthesis initiation.

Our approach is similar in concept to spatial 'omics methods. To quantify proteins, we will use recently developed optimized methods for the generation of stable-isotope mass spectrometry (MS) protein peptide mass standards. These mass standards are combined in known quantities with unlabelled biological samples derived from cell extracts treated to stabilise native interactions and fractionated into distinct complexes by gradient fractionation and/or association with a specific tagged factor. Analyses of the data enables precise quantification of individual protein levels. By also applying RNAseq to the same cell fractionated samples, we will quantify individual RNA levels in each fraction. In parallel, we will use the latest single molecule RNA microscopy methods with specific fluorescently labelled components to quantify translation of individual selected exemplar mRNAs in vivo both in actively growing cells and those subjected to acute stress, specific mutation or inhibitor compound treatment.