Human G3BP1 interacts with beta-F1-ATPase mRNA and inhibits its translation.

The post-transcriptional regulation of nuclear mRNAs that encode core components of mitochondria has relevant implications in cell physiology. The mRNA that encodes the catalytic subunit of the mitochondrial H(+)-ATP synthase subunit beta (ATP5B, beta-F1-ATPase) is localized in a large ribonucleoprotein (RNP) complex (beta-F1-RNP), which is subjected to stringent translational control during development and the cell cycle, and in carcinogenesis. Because downregulation of beta-F1-ATPase is a conserved feature of most prevalent human carcinomas, we have investigated the molecular composition of the human beta-F1-RNP. By means of an improved affinity-chromatography procedure and protein sequencing we have identified nine RNA-binding proteins (RNABPs) of the beta-F1-RNP. Immunoprecipitation assays of Ras-GAP SH3 binding protein 1 (G3BP1) and fluorescent in-situ hybridization of mRNA indicate a direct interaction of the endogenous G3BP1 with mRNA of beta-F1-ATPase (beta-F1 mRNA). RNA-bridged trimolecular fluorescence complementation (TriFC) assays confirm the interaction of G3BP1 with the 3'-UTR of beta-F1 mRNA in cytoplasmic RNA-granules. Confocal and high-resolution immunoelectron-microscopy experiments suggest that the beta-F1-RNP is sorted to the periphery of mitochondria. Molecular and functional studies indicate that the interaction of G3BP1 with beta-F1 mRNA inhibits its translation at the initiation level, supporting a role for G3BP1 in the glycolytic switch that occurs in cancer.

HuR and the bioenergetic signature of breast cancer: a low tumor expression of the RNA-binding protein predicts a higher risk of disease recurrence.

Downregulation of the catalytic subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) is a hallmark of many types of cancer. The expression of beta-F1-ATPase is stringently controlled by posttranscriptional mechanisms. Herein, we pursue the identification of beta-F1-ATPase messenger RNA-binding proteins (beta-mRNABPs) that interact and could define the bioenergetic phenotype of the cancer cell in order to establish its relevance as markers of breast cancer progression. RNA immunoprecipitation and RNA affinity chromatography identify HuR as a beta-mRNABP that interacts with the 3'-untranslated region of the transcript. Subcellular fractionation and high-resolution immunoelectron microscopy revealed the cofractionation and presence of HuR in subcellular structures associated to liver mitochondria. Analysis of the expression level of HuR in a cohort of breast carcinomas shows its association with the degree of alteration of the bioenergetic phenotype of the tumor. Moreover, HuR expression is shown to be an independent marker of breast cancer prognosis. A low tumor expression of HuR predicts a higher risk of disease recurrence in early stage breast cancer patients as assessed by clinical and bioenergetic markers of prognosis, strongly supporting the incorporation of HuR as an additional marker for the follow-up of these patients. Mechanistically, overexpression experiments and short hairpin RNA-mediated silencing of HuR in human embryonic kidney and HeLa cells indicate that HuR is not regulating beta-F1-ATPase expression. Overall, the participation of additional RNA-binding proteins in controlling beta-F1-ATPase expression and therefore in defining the bioenergetic signature of the cancer cell is expected.

A message emerging from development: the repression of mitochondrial beta-F1-ATPase expression in cancer.

Mitochondrial research has experienced a considerable boost during the last decade because organelle malfunctioning is in the genesis and/or progression of a vast array of human pathologies including cancer. The renaissance of mitochondria in the cancer field has been promoted by two main facts: (1) the molecular and functional integration of mitochondrial bioenergetics with the execution of cell death and (2) the implementation of (18)FDG-PET for imaging and staging of tumors in clinical practice. The latter, represents the bed-side translational development of the metabolic hallmark that describes the bioenergetic phenotype of most cancer cells as originally predicted at the beginning of previous century by Otto Warburg. In this minireview we will briefly summarize how the study of energy metabolism during liver development forced our encounter with Warburg's postulates and prompted us to study the mechanisms that regulate the biogenesis of mitochondria in the cancer cell.

Permeability properties of a three-cell type in vitro model of blood-brain barrier.

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood-brain barrier (BBB) properties if co-cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of 3H-sucrose able to cross the BCEC layer in this three-cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decreases even more. By Western analysis, we also found that, in addition to the canonical 60 kDa occludin, anti-occludin antibodies recognize a smaller protein of 48 kDa which accumulates during rat brain development. Interestingly this latter protein is present at higher amounts in endothelial cells cultured in the presence of both astrocytes and neurons, that is in those conditions in which sucrose permeation studies indicate formation of BBB.