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Many recent studies established a potential role for p53 in translational control of many transcripts through the modulation of the expression of several microRNAs, RNA binding proteins (RBPs), translation factors, or of ribosome biogenesis factors. To establish the relevance of translation control in p53-dependent apoptosis our group recently compared by polysome profiling two cell lines, SJSA1 and HCT116, undergoing respectively p53-dependent apoptosis and cell cycle arrest in response to the treatment with Nutlin-3, a selective activator of p53. Examining the markedly different, treatment-induced translation landscapes, the RNA helicase DHX30 was identified as a protein able to bind only in HCT116 cells to a 3’-UTR sequence motif, labeled as CGPD-motif. The motif had been identified as highly over-represented among translationally enhanced mRNAs in nutlin-treated SJSA1 cells, among which apoptosis effectors were enriched. Since the binding of DHX30 to the CGPD-motif transcripts is observed in cell cycle arresting HCT116, where those mRNA are not translationally enhanced, we reasoned that DHX30 could be part of an RBP complex responsible for reduced translation of specific pro-apoptotic mRNAs. To pursue this hypothesis, HCT116-shDHX30 clones were obtained and I studied the functions of this relatively unknown RNA helicase in this and other cell models. A translatome analysis by RNA-seq of HCT116-shDHX30 cells showed higher activation of ribosome biogenesis pathways, according to GSEA. Consistently, eCLIP data from ENCODE indicate that DHX30 can bind to most ribosomal protein transcripts, and those showed higher relative translation efficiency in HCT116-shDHX30 cells. In fact, the cells exhibited increased rRNA synthesis and higher global translation as well as a large expansion of the number of transcripts showing higher polysome-association in response to Nutlin. The DHX30 gene features two promoters, one of which can produce a transcript containing a mitochondrial localization signal. I confirmed that DHX30 has both a cytoplasmic and a mitochondrial localization by both cell fractionation and immunofluorescence. HCT116_shDHX30 cells have reduced mitochondrial metabolism, based on oxygen consumption rates but do not show evidence of compensatory glycolytic activity. Impaired mitochondrial functions could be related also to decreased expression of mitochondrial oxidative phosphorylation (OXPHOS) components as well as reduced expression of nuclear encoded mitoribosome proteins. Based on RIP assays DHX30 appears to target directly mitoribosome transcripts. Depletion of DHX30 showed reduced proliferation in various assays both in 2D and 3D culture conditions and sensitized cells to the treatment with the mitochondrial uncoupler FCCP. A similar role was observed also on mitochondrially encoded OXPHOS compomenents as well as mitoribosome components in MCF7 breast cancer cell line silenced for DHX30 with a consistent reduction in cell proliferation. In contrast, U2OS osteosarcoma cells did not show a change in the expression in mitochondrial components as well as an impact on proliferation after transient DHX30 silencing. Hence, it appears that DHX30 can exert a broad general control on cell metabolism and translation as well as a cis-element mediated control of the translation of specific mRNAs impacting both on cell fitness and apoptosis.

Mitochondrial DNA (mtDNA) is an essential component of mitochondria and consists of 13 protein coding genes which encode 13 subunits of the oxidative phosphorylation (OXPHOS) machinery. According to widely accepted theory, the mitochondrion was derived from an alpha-proteobacteria which was engulfed by an early eukaryotic cell and eventually evolved into the organelle through a process called endosymbiosis. Most of the proteins located in mitochondria are encoded by nuclear genes. Through the endosymbiotic process most of the bacterial genes were transferred to the nucleus leaving a small genome inside mitochondria (mtDNA). Looking at the DNA replication machinery of mtDNA, the components share structural similarity with viral replication machinery making it vulnerable to drugs targeted for antiviral therapy. Additionally, mitochondrion is one of the primary sources of cellular reactive oxygen species (ROS) production. Different chemical compounds and environmental toxicants can disrupt mitochondrial function resulting in increased ROS production leading to mitochondrial damage. Damaged mitochondria are detrimental to cellular health as the ROS produced by mitochondria can damage other cellular components. To protect the cell from damaged mitochondria, a specialized autophagy process is required called mitophagy which is responsible for clearing damaged mitochondria through the autophagic pathway. Moreover, damaged mitochondria can activate apoptotic signaling leading to cell death. To investigate the role of replication machinery on mitochondrial function, we utilized a cell line harboring a mutation in the POLG gene leading to a change in the amino acid position 955 from Tyr to Cys (Y955C) situated at the active site of the mitochondrial DNA polymerase, Polγ (chapter 2). We observed reduced growth rate, reduced mtDNA copy number, and increased sensitivity to the mtDNA toxicant dideoxycytidine (ddC) in the variant cell line relative to control. Additionally, there were reduced mtDNA nucleoids which support decreased mtDNA copy number. One and two-dimensional agarose gel electrophoresis analysis showed an increased abundance of Y955C mtDNA replication intermediates and linear molecules which likely result from double strand breaks due to replication stress. Moreover, the mtDNA encoded complex I subunit, NADH dehydrogenase subunit 3 and nuclear DNA encoded complex I subunit, NADH:ubiquinone oxidoreductase subunit B8 protein levels decreased in the variant cell line compared to control which indicates OXPHOS complex I activity and assembly is affected by the Y955C mutation. This indicates that dysfunction in mtDNA replication can affect mitochondrial function and cellular growth. Phthalates are a class of chemical compounds which are used as plasticizers. During the use of plastics, phthalates can leach out from the plastic and go inside the body. The detrimental effect of phthalate toxicity on mitochondrial function is reported in chapter 3 & 4. We explored the clearance of damaged mitochondria through PINK1-PARKIN pathway during MEHP exposure. MEHP exposure also affects mtDNA copy number in undifferentiated HepaRG cells. Also, one of the essential components of mtDNA replisome, MGME1 could be affected by MEHP exposure. This indicates that MEHP toxicity affects mitochondrial function and mtDNA copy number leading to mitochondrial damage which is likely cleared by the autophagy pathway. As mitochondria play an important role in apoptosis, the effect of MEHP exposure on the induction of cell death was assessed. During MEHP exposure, apoptotic cell death was observed, and apoptotic factors were induced. This indicate that acute MEHP toxicity disrupt mitochondrial function leading to autophagic clearance of mitochondria and induction of apoptotic cell death. Our observations explored the role of mitochondrial replication machinery in maintaining mitochondrial health and how disruption of mtDNA replication can affect mtDNA maintenance and cellular growth. Also, the toxic effect of MEHP exposure on mitochondrial function was observed which is evident by induction of mitophagy and apoptosis during exposure of MEHP. Insights from these studies help us to understand the mechanism of mitochondrial disease related to mutations in the mtDNA replication machinery and the consequence of environmental toxicant exposure on mitochondrial and cellular fate.

[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.