Molecular mechanisms controlling the translation of the mrna encoding the human catalytic beta-subunit of mitochondrial h+-atp synthase in cancer an development

Resumen

Mitochondria are essential organelles in cell physiology, playing key roles in bioenergetics, the execution of the cell death and intracellular signaling by Ca2+ and reactive oxygen species (ROS). Mitochondrial dysfunction is associated with a large number of human pathologies, which include cancer, diabetes and neurodegeneration. It is nowadays accepted that a phenotypic trait of most human carcinomas is the reprogramming of their cellular energetic metabolism from mitochondrial oxidative phosphorylation to an enhanced aerobic glycolysis, the so-called Warburg Effect. In this scenario, the mitochondrial H+-ATP synthase is an essential component both in the transduction of biological energy as well as in the execution of cell death. In fact, the down-regulation of the catalytic subunit of the H+-ATP synthase (ß-F1-ATPase) is a hallmark of many human carcinomas affording a marker of prognosis and of the response to therapy. Moreover, cancer cells and tumors over-express IF1, an inhibitor of the H+-ATP synthase. In the present PhD Thesis we have investigated the molecular mechanisms that control at post-transcriptional levels the expression of human ß-F1-ATPase. We illustrate that down-regulation of ß-F1-ATPase in human breast, lung, esophageal and colon cancer originates from a specific translation repression event that can be recapitulated in in vitro translation assays. We demonstrate that the human 3¿UTR of ß-F1-ATPase mRNA (ß-mRNA) is an important cis-acting element necessary for efficient translation. However, and at variance with previous findings with the rat transcript, recapitulation of translational repression requires the participation of additional cis-acting elements of the human transcript. In order to characterize the molecular mechanisms that underlie the masking of ß-mRNA we have undertaken studies aimed at the identification of the RNA binding proteins and miRNAs that target the human transcript. Herein, we demonstrate that Ras-GAP SH3 binding protein 1 (G3BP1) interacts within the cellular context with the 3¿UTR of ß-mRNA and is part of the cytoplasmic RNA granules that contain ß-mRNA. Furthermore, we show that G3BP1 promotes the repression of ß-mRNA translation both in vivo and in vitro. Specifically, we demonstrate that G3BP1 hampers the initiation step of ß-mRNA translation, strongly supporting that the regulated binding of G3BP1 to the transcript might be involved in masking the translation of ß-mRNA in human cancer. Moreover, we demonstrate in a cohort of 93 breast cancer patients that a high expression of G3BP1 affords a marker of cancer progression, specially providing a reliable indicator of developing metastatic disease within the group of breast cancer patients with a good prognosis as assessed by their metabolic phenotype. In contrast to these findings, we show that the RNABPs IMP1 and NPM1 do not interact with ß-mRNA playing no relevant role in ß-F1-ATPase biology. Finally, we have developed cellular systems to analyze the role of miRNAs in ß-F1-ATPase expression. Using these systems we show that ß-mRNA is targeted and translationally silenced by miR-127-5p, whereas miR-101, miR-103, miR-186, miR-200b, miR-423-5p and miR-581 have no apparent functional role. miR-127-5p is not expressed in human cancer cell lines. However, we observed its expression in human fetal liver strongly suggesting that it might play a relevant role controlling the translation of ß-mRNA during development of the human liver.