Molecular biology of octocoral mitochondria

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.