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While we have learned that mRNA sequence strongly influences translation and co-translational pathways, the exact mechanisms by which this sequence and the RNA structures encoded impact these steps of gene expression are less understood. Therefore, more investigations are needed to reveal how translation elongation and efficiency is influenced by RNA sequence and how the affected translation regulates and determines a variety of co-translational pathways. In this dissertation, I explore the effects of translational kinetics on a series of co-translational pathways using the budding yeast Saccharomyces cerevisiae as a model organism. In Chapter 2, I developed an in-vivo elongation reporter in Saccharomyces cerevisiae to quantitatively monitor translation elongation duration and protein expression. Using this elongation reporter, I investigated the effects of elongation stalls induced by different types of genetic factors on gene expression and demonstrated that distinct ribosomal stalls may trigger distinct co-translational pathways. In Chapter 3, I studied co-translational mRNA localization to mitochondrion, and proposed that translational kinetics, such as ribosomal stall caused by polyprolines, play an important role in mediating co-translational import. In addition, I further studied the effects of elongation stalls on mitochondrial import stress and triggering of relevant quality control pathways. In Chapter 4, I investigated the mechanism of cytosolic mRNP granule formation under glucose deprivation condition. In this study, I use CRISPRi to knockdown expression of RVB2 and confirm its essential role in deciding the fate of mRNA localization and translatability after glucose depletion. Finally in Chapter 5, I address the enhancements made to the developed method, outline potential future directions for the research presented in this dissertation, and conclude with my final remarks.

Mitochondria are involved in many essential cell functions, including the production of energy and cellular metabolism. Hence, a better understanding of how mitochondria adapt to different interventions may have implications for both health and performance. The ability to distinguish fibre-specific changes may help resolve some of the debate concerning the effects of different types of exercise on mitochondrial biogenesis. Skeletal muscle fibres represent a large proportion of cell types in humans, with specialised contractile and metabolic functions that depend on a large number of associated proteins with extensive posttranslational modifications. Many of these skeletal muscle proteins are present in a cell-specific or a fibre-type dependent manner. However, while the size principle has clearly demonstrated the recruitment of different fibre types with exercise of different intensities, the methods for studying mitochondrial protein adaptations have mostly been confined to the analysis for whole-muscle samples that contain a mixture of type I and II fibres. Recent advances in proteomics by mass spectrometry (MS) allow for the quantification of thousands of proteins in small biological samples, providing the potential to analyse changes in mitochondrial proteins in single muscle fibres. This research describes a proteomic workflow for fibre typing and the subsequent identification of mitochondrial proteins within single human skeletal muscle fibres by MS, even when fibres have been prepared in a high detergent matrix. Fibre types were verified based on the relative abundance of myosin heavy chain (MYH) isoforms, which was determined by dividing the intensity-based absolute quantification of the respective isoform (MYH1, MYH2, MYH4, MYH7) by the sum of the intensities of all four MYH isoforms. This protocol also allows for incorporation of tandem mass tag (TMT) labelling for increased identification of lowly abundant proteins using a TMTpro-16 plex. This permitted a three-tiered comparison of mitochondrial protein content from different fibre types applied to a post vs. pre exercise training study design comparing two different types of exercise training performed by 23 men. The developed proteomic workflow quantified the levels of 536 known mitochondrial proteins, representing more than 45% of the total mitochondrial proteins in single muscle fibres. Analysis of proteins associated with known cellular pathways within the mitochondria demonstrated distinct trends of fibre-type specific protein responses to different types of exercise. This research aims to further the application of proteomic technologies to better understand how specific mitochondrial proteins are altered in response to the stress of exercise at the resolution of single skeletal muscle fibres.