José M. Izquierdo

Mitochondrial Biogenesis in the Liver during Development and Oncogenesis

The analysis of the expression of oxidative phosphorylation genes in the liver during development reveals the existence of two biological programs involved in the biogenesis of mitochondria. Differentiation is a short-term program of biogenesis that is controlled at post-transcriptional levels of gene expression and is responsible for the rapid changes in the bioenergetic phenotype of mitochondria. In contrast, proliferation is a long-term program controlled both at the transcriptional and post-transcriptional levels of gene expression and is responsible for the increase in mitochondrial mass in the hepatocyte. Recently, a specific subcellular structure involved in the localization and control of the translation of the mRNA encoding the β-catalytic subunit of the H+-ATP synthase (β-mRNA) has been identified. It is suggested that this structure plays a prominent role in the control of mitochondrial biogenesis at post-transcriptional levels. The fetal liver has many phenotypic manifestations in common with highly glycolytic tumor cells. In addition, both have a low mitochondrial content despite a paradoxical increase in the cellular representation of oxidative phosphorylation transcripts. Based on the paradigm provided by the fetal liver we hypothesize that the aberrant mitochondrial phenotype of fast-growing hepatomas represents a reversion to a fetal program of expression of oxidative phosphorylation genes by the activation, or increased expression, of an inhibitor of β-mRNA translation.

RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage

Cytopathic viruses have developed successful strategies to block or, at least, to attenuate host interference with their replication. Here, we have analyzed the effects of poliovirus 2A protease on RNA nuclear export. 2A protease interferes with trafficking of mRNAs, rRNAs and U snRNAs from the nucleus to the cytoplasm, without any apparent effect on tRNA transport. Traffic of newly produced mRNAs is more strongly affected than traffic of other mRNAs over-represented in the cytoplasm, such as mRNA encoding β-actin. Inhibition of RNA nuclear export in HeLa cells expressing 2A protease is concomitant with the cleavage of Nup98, Nup153, Nup62 and their subsequent subcellular redistribution. The expression of an inactive 2A protease failed to interfere with RNA nuclear export. In addition, other related proteases, such as poliovirus 3C or foot and mouth disease virus Lpro did not affect mRNA distribution or Nup98 integrity. Treatment of HeLa cells with interferon (IFN)-γ increased the relative amount of Nup98. Under such conditions, the cleavage of Nup98 induced by 2A protease is partial, and thus IFN-γ prevents the inhibition of RNA nuclear export. Taken together, these results are consistent with a specific proteolysis of Nup98 by 2A protease to prevent de novo mRNA traffic in poliovirus-infected cells.

A conserved mechanism for controlling the translation of beta-F1-ATPase mRNA between the fetal liver and cancer cells.

To characterize the mechanisms governing the biogenesis of mitochondria in cancer, we studied the mitochondrial phenotype and the mechanisms controlling the expression of the β subunit of the mitochondrial H+-ATP synthase (β-F1-ATPase) gene in the rat FAO and AS30D hepatomas. When compared with normal adult rat liver, the relative cellular content of the mitochondrial β-F1-ATPase and glutamate dehydrogenase, as well as of mitochondrial DNA, was severely reduced in both cell lines. A paradoxical increase in the cellular abundance of β-F1-ATPase mRNA was observed in cancer cells. Run-on transcription assays and the estimation of mRNA half-lives revealed that the increased abundance of β-F1-ATPase mRNA results from the stabilization of the transcript in cancer. In vitro translation assays revealed a specific inhibition of the synthesis of the β-precursor when translation reactions were carried out in the presence of extracts derived from cancer cells. The inhibitory effect was recapitulated using an RNA chimera that contained the 3′-untranslated region of β-F1-ATPase mRNA. Hepatoma extracts also contained an increased activity of the developmentally regulated translation-inhibitory proteins that bind the 3′-untranslated region of β-F1-ATPase mRNA. The results indicate that the expression of this gene in hepatoma cells is controlled by the same mechanisms that regulate its expression in the liver during fetal development.

Depletion of T-cell intracellular antigen proteins promotes cell proliferation

BackgroundT-cell intracellular antigen-1 (TIA-1) and TIA-1 related/like protein (TIAR/TIAL1), two DNA/RNA binding proteins broadly expressed in eukaryotic cells, participate in the regulation of gene expression through RNA metabolism. Despite the biological relevance of these regulators, there are no genome-wide studies assessing global transcriptomic and phenotypic impacts after changes in the expression and/or function of these proteins.ResultsUsing high-throughput gene expression profiling, we found that the TIA-1/TIAR-depleted cell phenotype is linked to a transcriptome involved in the control of inflammation, cell-cell signaling, immune-suppression, angiogenesis, metabolism and cell proliferation. Induced genes included pro-inflammatory cytokines, inflammatory chemokines, growth-stimulating factors and pro-angiogenic inducers. Repressed genes involved the RAS oncogene family member RAB40B, regulators of cytoskeleton organization and biogenesis and a mitochondrial modulator. Consistent with these observations, depletion of TIA proteins in HeLa cells results in increased cell proliferation, altered cell-cycle and anchorage-independent growth. Mechanistically, the changes associated with the steady-state target mRNA levels regulated by TIA proteins are consistent with overlapping effects on gene basal transcription rate and mRNA turnover.ConclusionsCollectively, our findings suggest a role for TIA proteins as cellular sensors that modulate gene expression control at the transcriptional and post-transcriptional levels, coupling cell proliferation responses and metabolic homeostasis to cell survival and growth.

Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth.

TIA (T-cell intracellular antigen) proteins function as DNA/RNA trans-acting regulators to expand transcriptome and proteome diversity in mammals. In the present paper we report that the stable silencing of TIA1 and/or TIAR/TIAL1 (TIA1-related/like protein 1) expression in HeLa cells enhances cell proliferation, anchorage-dependent and -independent growth and invasion. HeLa cells lacking TIA1 and/or TIAR generate larger and faster-growing epithelial tumours with high rates of proliferation and angiogenesis in nude mice xenografts. Protein array analysis of a collection of human tumours shows that TIA1 and TIAR protein expression is down-regulated in a subset of epithelial tumours relative to normal tissues. Our results suggest a link between the epigenetic control exerted by TIA proteins and the transcriptional and post-transcriptional regulation of a subset of specific genes involved in tumour progression. Taken together, these results are consistent with a role for TIA proteins as growth/tumour-suppressor genes.

Cell-specific regulation of Fas exon 6 splicing mediated by Hu antigen R.

The differential expression levels of T-cell intracellular antigens (TIA) and Hu antigen R (HuR) are concomitant with a splicing switch in apoptosis receptor Fas in HCT-116 cells. Thus, overexpression and knockdown of HuR led to Fas exon 6 skipping and inclusion, respectively. These results suggest that the TIA and HuR cellular ratio influences cell-type specific Fas exon 6 splicing pattern.

Poliovirus 2A Protease Triggers a Selective Nucleo- Cytoplasmic Redistribution of Splicing Factors to Regulate Alternative Pre-mRNA Splicing

Poliovirus protease 2A (2Apro) obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2Apro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2Apro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2Apro-expressing cells. Therefore, poliovirus 2Apro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

T-cell intracellular antigen (TIA)-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy.

Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress.

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling.

BackgroundT-cell intracellular antigen (TIA) proteins function as regulators of cell homeostasis. These proteins control gene expression globally at multiple levels in response to dynamic regulatory changes and environmental stresses. Herein we identified a micro(mi)RNA signature associated to transiently TIA-depleted HeLa cells and analyzed the potential role of miRNAs combining genome-wide analysis data on mRNA and miRNA profiles.ResultsUsing high-throughput miRNA expression profiling, transient depletion of TIA-proteins in HeLa cells was observed to promote significant and reproducible changes affecting to a pool of up-regulated miRNAs involving miR-30b-3p, miR125a-3p, miR-193a-5p, miR-197-3p, miR-203a, miR-210, miR-371-5p, miR-373-5p, miR-483-5p, miR-492, miR-498, miR-503-5p, miR-572, miR-586, miR-612, miR-615-3p, miR-623, miR-625-5p, miR-629-5p, miR-638, miR-658, miR-663a, miR-671-5p, miR-769-3p and miR-744-5p. Some up-regulated and unchanged miRNAs were validated and previous results confirmed by reverse transcription and real time PCR. By target prediction of the miRNAs and combined analysis of the genome-wide expression profiles identified in TIA-depleted HeLa cells, we detected connections between up-regulated miRNAs and potential target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis suggest that target genes are related with biological processes associated to the regulation of DNA-dependent transcription, signal transduction and multicellular organismal development as well as with the enrichment of pathways involved in cancer, focal adhesion, regulation of actin cytoskeleton, endocytosis and MAPK and Wnt signaling pathways, respectively. When the collection of experimentally defined differentially expressed genes in TIA-depleted HeLa cells was intersected with potential target genes only 7 out of 68 (10%) up- and 71 out of 328 (22%) down-regulated genes were shared. GO and KEGG database analyses showed that the enrichment categories of biological processes and cellular pathways were related with innate immune response, signal transduction, response to interleukin-1, glomerular basement membrane development as well as neuroactive ligand-receptor interaction, endocytosis, lysosomes and apoptosis, respectively.ConclusionAll this considered, these observations suggest that individual miRNAs could act as potential mediators of the epigenetic switch linking transcriptomic dynamics and cell phenotypes mediated by TIA proteins.