The Roles of Mitochondrial Protein Synthesis and of the Transcriptional Coactivator Spargel in Drosophila Growth and Development

    Tutkimustuotos: VäitöskirjaCollection of Articles

    Abstrakti

    When the capacity of mitochondrial ribosomes (mitoribosomes) to translate genes encoded in the mitochondrial genome (mtDNA) is impaired, severe effects on mitochondrial activity and, subsequently, on total cellular metabolism, result. The impacts can be observed throughout the entire organism and present as a metabolic disease. In humans, such disease is often seen when mutations arise in nuclearly or mitochondrially encoded components of the mitoribosome or its associated molecular machinery. Understanding the mechanistic pathways of these diseases and exploring methods to alleviate their symptoms requires an intricate understanding of the metabolic changes that underpin these pathologies.

    The work presented within this thesis aimed at adding to this understanding by using genetic and chemical alterations to scrutinise the complex context of metabolism during development in a Drosophila melanogaster mutant model of mitoribosomal disease. The Drosophila strain tko25t, carrying a mis-sense mutation in mitoribosomal small subunit protein S12 (mRpS12), has phenotypes including bang sensitivity, impaired hearing and developmental delay, closely phenocopying elements of human mitochondrial disease. The same is true at the cellular and molecular level where a decreased capacity for mitochondrial protein synthesis causes tko25t to exhibit decreased electron transport chain (ETC) complex activity, a marker of mitochondrial disease in humans.

    Using a combination of transcriptomic and metabolomic assays, along with genetic perturbations, dietary manipulations and developmental analyses, a sensitivity to alterations in pyruvate metabolism was identified as a key component of the tko25t mutant phenotype, in which pyruvate levels were found to be basally higher than in wild-type flies. Adding pyruvate to the diet of tko25t or decreasing the expression of crucial components of pyruvate metabolism led to significant changes in growth rate and provided insight into the metabolic effects of decreased mitoribosomal activity. Altered pyruvate metabolism was closely linked to an inferred compensation mechanism in tko25t mutant flies, whereby glucose serum levels were lowered through changes in the expression of putative Malpighian tubule- and gut-specific sugar transporters. By lowering the sugar level of the culture medium, which resulted in a decreased level of serum sugar, key features of the tko25t mutant phenotype were alleviated; notably the extent of developmental delay and the recovery time from mechanical shock (bang sensitivity), consistent with a suggested ‘sugar-toxicity’ toward the mitoribosomal mutant flies.

    To further explore whether the tko25t phenotype could be alleviated through alterations at the genetic rather than the metabolic level, the transcriptional coactivator spargel was over-expressed. spargel belongs to the PGC1 family of coactivators that have been proposed as master regulators of mitochondrial biogenesis. In the adult Drosophila fat body, spargel has been shown to alter mitochondrial transcript expression. Additionally, the expression of spargel at the RNA level is decreased in the tko25t mutant. It was therefore reasoned that spargel overexpression might correct the decreased capacity for mitochondrial protein synthesis in tko25t mutant flies, and thus alleviate its developmental delay accordingly. However, spargel overexpression did not have the intended outcome of increased mitochondrial gene transcription and biogenesis in the tko25t mutant, nor in control flies.

    Whilst carrying out these experiments, spargel RNA expression in adult Drosophila females was observed to be approximately ten-fold higher than in males. Further analysis found this to be a result of spargel RNA levels being high in ovaries and remaining high throughout the earliest stages of embryogenesis. To gain further insight into the biological function(s) of spargel, its expression was knocked down at the RNA level, specifically in the female germline. The embryonic semi-lethality that resulted confirmed the essential nature of spargel in the developing Drosophila embryo. Experiments were undertaken to determine whether spargel, despite its lack of interaction with tko25t, was functioning to boost mitochondrial biogenesis during these early stages of development. This also failed to find any evidence linking spargel activity with mitochondrial regulation. These findings confirm the importance of spargel function in development, but call into question its often-assumed role in mitochondrial biogenesis.
    AlkuperäiskieliEnglanti
    JulkaisupaikkaTampere
    KustantajaTampere University
    ISBN (elektroninen)978-952-03-1667-9
    ISBN (painettu)978-952-03-1666-2
    TilaJulkaistu - 2020
    OKM-julkaisutyyppiG5 Artikkeliväitöskirja

    Julkaisusarja

    NimiTampere University Dissertations - Tampereen yliopiston väitöskirjat
    Vuosikerta296
    ISSN (painettu)2489-9860
    ISSN (elektroninen)2490-0028

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