Genome-wide profiling reveals a role for T-cell intracellular antigens TIA1 and TIAR in the control of translational specificity in HeLa cells.

TIA (T-cell intracellular antigens)-knockdown HeLa cells show an increase in ribosomes and translational machinery components. This increase correlates with specific changes in translationally up-regulated mRNAs involved in cell-cycle progression and DNA repair, as shown in polysomal profiling analysis. Our data support the hypothesis that a concerted activation of both global and selective translational rates leads to the transition to a more proliferative status in TIA-knockdown HeLa cells.

Long-term reduction of T-cell intracellular antigens leads to increased beta-actin expression.

BACKGROUND: The permanent down-regulated expression of T-cell intracellular antigen (TIA) proteins in HeLa cells improves cytoskeleton-mediated functions such as cell proliferation and tumor growth. METHODS: Making use of human and mouse cells with knocked down/out expression of T-cell intracellular antigen 1 (TIA1) and/or TIA1 related/like (TIAR/TIAL1) proteins and classical RNA (e.g. reverse transcription-quantitative polymerase chain reaction, polysomal profiling analysis using sucrose gradients, immunoblotting, immunoprecipitation, electrophoretic mobility shift assays, ultraviolet light crosslinking and poly (A+) test analysis) and cellular (e.g. immunofluorescence microscopy and quimeric mRNA transfections) biology methods, we have analyzed the regulatory role of TIA proteins in the post-transcriptional modulation of beta-actin (ACTB) mRNA. RESULTS: Our observations show that the acquisition of above cellular capacities is concomitant with increased expression levels of the actin beta subunit (ACTB) protein. Regulating TIA abundance does not modify ACTB mRNA levels, however, an increase of ACTB mRNA translation is observed. This regulatory capacity of TIA proteins is linked to the ACTB mRNA 3'-untranslated region (3'-UTR), where these proteins could function as RNA binding proteins. The expression of GFP from a chimeric reporter containing human ΑCΤΒ 3'-UTR recapitulates the translational control found by the endogenous ACTB mRNA in the absence of TIA proteins. Additionally, murine embryonic fibroblasts (MEF) knocked out for TIA1 rise mouse ACTB protein expression compared to the controls. Once again steady-state levels of mouse ACTB mRNA remained unchanged. CONCLUSIONS: Collectively, these results suggest that TIA proteins can function as long-term regulators of the ACTB mRNA metabolism in mouse and human cells.

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

BACKGROUND: T-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. RESULTS: Using 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. CONCLUSION: All 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.

Evidence of ongoing replication in a human immunodeficiency virus type 1 persistently infected cell line.

Human immunodeficiency virus type 1 (HIV-1) persistently infected cell lines are characterized by the continuous viral production without cytopathic effect. However, it is not completely clear if this production is contributed only by viral transcription or also by new cycles of viral replication. We studied an HIV-1 persistently infected cell line, designated H61-D, providing evidence of new replication cycles as sustained by: (i) a decrease in viral production, measured by p24 protein, after treatment of the culture with 3'-azydo-3'-deoxythymydine; (ii) detection of new integration events in the course of cell culture, and (iii) finding of two-long-terminal repeat circles in the cells. H61-D cells were not infected by cell-free virus, but infection was possible by co-culture with another productive-infected cell line. In conclusion, ongoing viral replication is taking place in H61-D persistent cells and new infections are mediated by a cell-to-cell spread mechanism.

A Heterologous Cell Model for Studying the Role of T-Cell Intracellular Antigen 1 in Welander Distal Myopathy.

Welander distal myopathy (WDM) is a muscle dystrophy characterized by adult-onset distal muscle weakness, prevalently impacting the distal long extensors of the hands and feet. WDM is an autosomal dominant disorder caused by a missense mutation (c.1362G>A; p.E384K) in the TIA1 (T-cell intracellular antigen 1) gene, which encodes an RNA-binding protein basically required for the posttranscriptional regulation of RNAs. We have developed a heterologous cell model of WDM to study the molecular and cellular events associated with mutated TIA1 expression. Specifically, we analyzed how this mutation affects three regulatory functions mediated by TIA1: (i) control of alternative SMN2 (survival motor neuron 2) splicing; (ii) formation, assembly, and disassembly of stress granules; and (iii) mitochondrial dynamics and its consequences for mitophagy, autophagy, and apoptosis. Our results show that whereas WDM-associated TIA1 expression had only a mild effect on SMN2 splicing, it led to suboptimal adaptation to environmental stress, with exacerbated stress granule formation that was accompanied by mitochondrial dysfunction and autophagy. Overall, our observations indicate that some aspects of the cell phenotype seen in muscle of patients with WDM can be recapitulated by ectopic expression of WDM-TIA1 in embryonic kidney cells, highlighting the potential of this model to investigate the pathogenesis of this degenerative disease and possible therapeutics.

T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression.

Mitochondria undergo frequent morphological changes to control their function. We show here that T-cell intracellular antigens (TIA1b/TIARb) and Hu antigen R (HuR) have antagonistic roles in mitochondrial function by modulating the expression of mitochondrial shaping proteins. Expression of TIA1b/TIARb alters the mitochondrial dynamic network by enhancing fission and clustering, which is accompanied by a decrease in respiration. In contrast, HuR expression promotes fusion and cristae remodeling and increases respiratory activity. Mechanistically, TIA proteins downregulate the expression of optic atrophy 1 (OPA1) protein via switching of the splicing patterns of OPA1 to facilitate the production of OPA1 variant 5 (OPA1v5). Conversely, HuR enhances the expression of OPA1 mRNA isoforms through increasing steady-state levels and targeting translational efficiency at the 3' untranslated region. Knockdown of TIA1/TIAR or HuR partially reversed the expression profile of OPA1, whereas knockdown of OPA1 or overexpression of OPA1v5 provoked mitochondrial clustering. Middle-term expression of TIA1b/TIARb triggers reactive oxygen species production and mitochondrial DNA damage, which is accompanied by mitophagy, autophagy, and apoptosis. In contrast, HuR expression promotes mitochondrion-dependent cell proliferation. Collectively, these results provide molecular insights into the antagonistic functions of TIA1b/TIARb and HuR in mitochondrial activity dynamics and suggest that their balance might contribute to mitochondrial physiopathology.

T-cell intracellular antigens in health and disease.

T-cell intracellular antigen 1 (TIA1) and TIA1-related/like protein (TIAR/TIAL1) are 2 proteins discovered in 1991 as components of cytotoxic T lymphocyte granules. They act in the nucleus as regulators of transcription and pre-mRNA splicing. In the cytoplasm, TIA1 and TIAR regulate and/or modulate the location, stability and/or translation of mRNAs. As knowledge of the different genes regulated by these proteins and the cellular/biological programs in which they are involved increases, it is evident that these antigens are key players in human physiology and pathology. This review will discuss the latest developments in the field, with physiopathological relevance, that point to novel roles for these regulators in the molecular and cell biology of higher eukaryotes.

Long-term reduction of T-cell intracellular antigens reveals a transcriptome associated with extracellular matrix and cell adhesion components.

Knockdown of T-cell intracellular antigens TIA1 and TIAR contributes to a cellular phenotype characterised by uncontrolled proliferation and tumorigenesis. Massive-scale poly(A+) RNA sequencing of TIA1 or TIAR-knocked down HeLa cells reveals transcriptome signatures comprising genes and functional categories potentially able to modulate several aspects of membrane dynamics associated with extracellular matrix and focal/cell adhesion events. The transcriptomic heterogeneity is the result of differentially expressed genes and RNA isoforms generated by alternative splicing and/or promoter usage. These results suggest a role for TIA proteins in the regulation and/or modulation of cellular homeostasis related to focal/cell adhesion, extracellular matrix and membrane and cytoskeleton dynamics.

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.

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells.

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2(')-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2(')-difluoro-2(')-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.