D
ISCUSSION
The data presented in this thesis has mainly focused on the characterisation of AURKAIP1, a protein about which very little data had been published at the beginning of my investigations. My aims at the outset and the progress I made towards achieving these aims are described below:
• To express and purify recombinant AURKAIP1 to use as an antigen for antibody production
I successfully overexpressed and purified sufficient recombinant mature AURKAIP1 to allow the production of custom made antisera. I affinity purified the antisera to reduce non-specific binding and characterised that the α- AURKAIP1 antibody was specific to AURKAIP1. Using this tool, I determined that the levels of endogenous AURKAIP1 were too low to be detected in 40µg of cell lysate, but could be identified in 50µg of isolated mitochondrial lysate from 143B cells.
• To produce stable human cell lines capable of expressing C-terminal FLAG- tagged AURKAIP1
I produced HEK293 cell lines that could inducibly overexpress AURKAIP1 with a C-terminal FLAG tag. I later identified that U2OS cells tolerated siRNA mediated AURKAIP1 depletion better and allowed improved visualisation of mitochondrial morphology by fluorescence microscopy compared to HEK293 cells. For these reasons, I also established U2OS lines capable of inducible AURKAIP1-FLAG expression.
• To use these tools to determine whether AURKAIP1 is mitochondrial, and if so, the sub-mitochondrial localisation
The α-AURKAIP1 antibody and AURKAIP1-FLAG expressing cell lines were used to show that AURKAIP1 is present in human mitochondria and is
• To characterise the phenotype of AURKAIP1 depletion, with a view to identifying any critical role of AURKAIP1 in mitochondrial gene expression
I have shown that AURKAIP1 is an essential protein required for cell viability and normal mitochondrial gene expression, as depletion of the protein causes mitochondrial translation defects.
• To identify binding partners of AURKAIP1 via immunoprecipitation to elucidate the potential functions of AURKAIP1
I have demonstrated a strong novel interaction between AURKAIP1 and p32 using Co-IP techniques. Weaker or more transient interactions were observed between AURKAIP1 and MRPs (MRPL12 and MRPL3), although further investigation would be required to demonstrate that these interactions were direct, rather than mediated by binding of each protein to p32.
Despite extensive study towards my original aims, the precise functional role of AURKAIP1 in human mitochondria remained elusive. One of the difficulties in determining the role of AURKAIP1 lies in the effects resulting from over or under- representational levels of AURKAIP1 protein. This was made apparent by the unexpected observation that AURKAIP1-FLAG overexpression as well as depletion led to an impairment of mitochondrial gene expression. AURKAIP1 depletion and AURKAIP1 overexpression each led to cellular phenotypes that included enlarged nucleoid morphology, reduced steady state levels of 12S rRNA, MT-CO1 mRNA and also of mtDNA-encoded proteins. Not all the changes were identical, a number of distinctions existed. AURKAIP1-FLAG overexpression caused a generalised defect in mitochondrial translation, whereas depletion of AURKAIP1 reduced the synthesis of only a specific subset of mtDNA-encoded proteins. Only AURKAIP1-FLAG overexpression led to a reduction in steady state levels of mitoribosomal subunits. This strongly suggests that separate signalling pathways underlie the mechanisms that cause the altered cellular phenotype following AURKAIP1 depletion or overexpression. The combination of overlapping and distinct differences in presentation mean that it is difficult to propose a hypothesis to explain all of the observed data at this stage.
It is not possible to say with certainty which of the observed effects following changes in steady state levels of AURKAIP1, are primary and which are secondary. The most striking effect of AURKAIP1-FLAG overexpression was the reduction in MRP levels. This was likely the result of disassembly of mitoribosomes since MRP levels were not reduced upon inhibition of mitoribosome biogenesis, over similar time frames (Dennerlein et al., 2010). The hypothesis that disassembly of the mitoribosome is the primary effect of AURKAIP1-FLAG overexpression offers the best explanation of the observed data, as this could lead to secondary effects of MRP degradation by the proteasome and subsequent impairment of mitochondrial protein synthesis due to the resulting lack of active mitoribosomes. In addition, the reduction in mitochondrial RNA levels may be explained by this hypothesis, as without the MRPs to protect the mRNA transcripts and rRNA, it is possible that they would be more rapidly degraded. Since my data shows that p32 depletion also reduces MRPL3 protein levels and published data has shown that loss of p32 causes a mitochondrial protein synthesis defect (Yagi et al., 2012), an attractive hypothesis is that AURKAIP1 acts to sequester p32 from its function. This may imply that p32 is involved in mitoribosome maintenance. That being said, p32 depletion and AURKAIP1-FLAG overexpression do not have identical phenotypes. This may be explained by p32 being a multifunctional protein. One explanation might be that AURKAIP1-FLAG overexpression blocks only one specific interaction of p32, leading to the impairment of protein synthesis, but other functions of p32 remain unaffected. In contrast p32 depletion would deprive the cells of all p32- mediated functions.
There are several other hypotheses, which may explain the data generated thus far, but further work is required to assess their likelihood. Of particular interest is assessing the nature of the interaction between AURKAIP1 and p32. Studies are currently on-going in my host laboratory, primarily focusing on determining:
• Whether AURKAIP1 can effect the relative amounts of p32 present as a trimer or hexamer and investigating the functional role of each
• Whether p32 and AURKAIP1 can form a complex that has protease activity • The crystal structure of AURKAIP1 both alone and in complex with p32.
The original aim of these investigations was to study the role of AURKAIP1 in mitochondrial gene expression. Unfortunately, the data described in this thesis is insufficient to conclusively elucidate this. If the effects of AURKAIP1-FLAG overexpression are found not to be mediated by p32, then perhaps studying AURKAIP1 depletion further may be appropriate. The most striking observation upon AURKAIP1 depletion is that synthesis of only a subset of mtDNA-encoded proteins is impaired. Perhaps AURKAIP1 acts to recruit specific mRNAs to the mitoribosome. Alternatively, certain transcripts may have a propensity to stall the mitoribosome during the elongation phase of translation and as such will not be recognised by mtRF1a and subsequently resolved. AURKAIP1 may act to rescue these stalled mitoribosomes, liberating the transcript allowing it to participate in another round of translation. If this were the case, then in AURKAIP1 depleted cells the mitoribosomes may remain stalled on this subset of specific transcripts, resulting in reduced synthesis of the encoded proteins. This hypothesis may also be able to account for the phenotype resulting from overexpression, as high levels of AURKAIP1 may result in a loss of specificity/selectivity and thus disassemble non-stalled mitoribosomes. These ideas are speculative and targeted experiments would be required to test the hypothesis. One approach that could be used to determine if there is a change in the stall profile is ribosome profiling. By performing this technique on mitoribosomes from AURKAIP1 depleted cells, any change in amount or position on transcripts of stall events could be detected and provide data that would challenge or substantiate the hypothesis.
Although the molecular mechanism by which AURKAIP1 contributes to mitochondrial gene expression is not yet clear, my studies have made a contribution to the increasing amounts of data concerning AURKAIP1. Since I have shown that both AURKAIP1 depletion and overexpression affect mitochondrial protein synthesis, I propose that AURKAIP1 plays a role in the regulation mitochondrial translation, potentially mediated though a titrated interaction with p32, however further work is required to confirm this. The identification of pathogenic mutations in p32, the precise function of which is also unknown, leading to mitochondrial disease (M.O. unpublished, personal
communication) serves to highlight the importance of understanding the fundamental
aspects of mitochondrial biology. Better comprehension of mitochondrial processes, in turn provides enhanced understanding of mitochondrial dysfunction, which could lead to advances in treatment strategies for patients suffering from mitochondrial disease.