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Shimpi, Gaurav Gokul (2015): Molecular biology of octocoral mitochondria: mtDNA repair, expression, and evolutionary implications. Dissertation, LMU München: Faculty of Geosciences



The mitochondria of non-bilaterian metazoans display a staggering diversity of genome organizations and also a slow rate of mtDNA evolution, unlike bilaterians, which may hold a key to understand the early evolution of the animal mitochondrion. Octocorals are unique members of Phylum Cnidaria, harboring several atypical mitochondrial genomic features, including a paucity of tRNA genes, various genome arrangements and the presence of novel putative mismatch repair gene (mtMutS) with various potential biological roles. Thus octocorals represents an interesting model for the study of mitochondrial biology and evolution. However, besides its utility in molecular phylogenetics, the mtDNA of octocorals is not studied from the perspective of DNA repair, oxidative stress response or gene expression; and there is a general lack of knowledge on the DNA repair capabilities and role of the mtMutS gene, response to climate-change, and mtDNA transcription in absence of interspersed tRNA genes of octocoral mitochondrial genome. In order to put the observed novelties in the octocoral mitochondria in an evolutionary and an environmental context, and to understand their potential functions and the consequences of their presence in conferring fitness during climate change induced stress, this study was undertaken. This dissertation aims to explore the uniqueness and diversity of octocoral mtDNA from an environmental as well as an evolutionary perspective. The thesis comprises five chapters exploring various facets of octocoral biology. The introductory section provides basic information and elaborates on the importance of studying non-bilaterian mitochondria. The first chapter sets the base for subsequent gene expression studies. Octocorals are extensively studied from a taxonomic and phylogenetic point of view. However, gene expression studies on these organisms have only recently started to appear. To successfully employ the most commonly used gene expression profiling technique i.e., the quantitation real-time PCR (qPCR), it is necessary to have an experimentally validated, treatment-specific set of stably expressed reference genes that will support for the accurate quantification of changes in expression of genes of interest. Hence, seven housekeeping genes, known to exhibit constitutive expression, were investigated for expression stability during simulated climate-changed (i.e. thermal and low-pH) induced stress. These genes were validated and subsequently used in gene expression studies on Sinularia cf. cruciata, our model octocoral. The occurrence of a mismatch repair gene, and the slow rates of mtDNA evolution in octocoral mitogenome calls for further investigations on the potential robustness of octocoral mitochondria to the increased oxidative stress. The second chapter presents a mitochondrion-centric view of climate-change stress response by investigating mtDNA damage, repair, and copy number dynamics during stress. The changes in gene expression of a set of stress-related nuclear, and mitochondrial genes in octocorals were also monitored. A robust response of octocoral mitochondria to oxidative mtDNA damage was observed, exhibiting a rapid recovery of the damaged mtDNA. The stress-specific regulation of the mtMutS gene was detected, indicating its potential involvement in stress response. The results highlight the resilience potential of octocoral mitochondria, and its adaptive benefits in changing oceans. The tRNA genes in animal mitochondria play a pivotal role in mt-mRNA processing and maturation. The influence of paucity of tRNA genes on transcription of the mitogenome in octocorals has not been investigated. The third chapter steps in the direction to understand the mitogenome transcription by investigating the nature of mature mRNAs. Several novel features not present in a “typical” animal mt-mRNAs were detected. The majority of the mitochondrial transcripts were observed as polycistronic units (i.e. the mRNA carrying information for the synthesis of more than one protein). 5’ and 3’ untranslated regions were delineated for most protein-coding genes. Alternative polyadenylation (APA) of mtMutS gene and long non-coding RNA (lncRNA) for ATP6 were detected and are reported for the first time in non-bilaterian metazoans providing a glimpse into the complexity and uniqueness of mtDNA transcription in octocorals. The mismatch repair (MMR) mechanism plays a crucial role in mutation avoidance and maintenance of genomic integrity. Its occurrence in animal mitochondria remains equivocal. Octocorals are the only known animals to posses an mtDNA-encoded MMR gene, the mtMutS, speculated to have self-contained DNA repair capability. In order to gain knowledge of the MMR activity in the octocoral mitochondria MMR assays using the octocoral mitochondrial fraction is necessary. A prerequisite for this assay is the availability of an MMR-substrate, which is a DNA fragment, usually a plasmid, containing the desired mismatch lesion (i.e. a heteroduplex) and a nicked strand. However, the methods to prepare such a substrate are time consuming and technically demanding. Chapter four describes two convenient and flexible strategies that can be used in parallel to prepare heteroduplex MMR substrate using a common plasmid and routine molecular biology techniques. This method should aid in MMR investigations in general, helping to advance this field of research. The mtMutS gene mentioned above is a bacterial homolog, predicted to have been horizontally transferred to the octocoral mitogenome. However, unlike the bacterial mutS, which is extensively studied, protein expression studies of the octocoral mtMutS gene are lacking. To investigate the biological role of the mtMutS protein, in vitro, and to gain knowledge on its structure and function, the expression of the gene in a bacterial host is necessary. The fifth chapter discusses the characteristics of the mtMutS protein, the efforts to express it in E. coli and some necessary precautions to be taken while working with the expression of such mtDNA-encoded proteins for the research in future. This dissertation elucidates and contributes to the understanding of the unexplored complexity of non-bilaterian mitochondria. It deals for the first time with DNA repair, gene expression and gene function, encompassing an integrative analysis of DNA, RNA and proteins to achieve its goals. This study forms the basis for many future investigations on the molecular mitochondrial biology of octocorals as well as other non-bilaterians, augmenting the understanding of the evolution of animal mitochondria, and also its role in cellular and organismal homeostasis in the context of environmental change.