Proteins T-cell Intracellular Antigen-1 Research Articles

A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence.

A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence

A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Unravelling the unfolding pathway of human Fas-activated serine/threonine kinase induced by urea

Fas-activated serine/threonine kinase (FASTK) is a mitochondria-associated nuclear protein that inhibits Fas- and UV-induced apoptosis. This protein is generally activated during Fas-mediated apoptosis by phosphorylating a nuclear RNA-binding protein T-cell intracellular antigen-1 and thus considered as a modulator of apoptosis. In the present study, we have examined the equilibrium unfolding and conformational stability of the kinase domain of FASTK (FASTK353-444 ). The kinase domain of FASTK353-444 was cloned, expressed, and purified. The folding ↔ unfolding transitions of urea-induced denaturation was monitored with the help of circular dichroism, intrinsic fluorescence, and UV absorption spectroscopies. Analysis of transition curves obtained from different probes revealed a coincidence of denaturation curves, suggesting that folding/unfolding of FASTK follows a two-state process with the midpoint (C m) value at 3.50 ± 0.1 M. Urea-induced denaturation curves were further analyzed to estimate change in the Gibbs free energy in the absence of urea (ΔG D 0) associated with the equilibrium of denaturation. To get atomistic insights into the urea-induced denaturation of FASTK, we performed an all-atom molecular dynamics simulation for 100 ns. A close agreement was noticed between experimental and computational studies. This study will help to understand the unfolding mechanism and structural stability of the kinase domain of FASTK. Communicated by Ramaswamy H. Sarma

Reduction of the RNA Binding Protein TIA1 Exacerbates Neuroinflammation in Tauopathy.

Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in tauopathies, a class of neurodegenerative disease characterized by aggregation of hyperphosphorylated tau protein. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however, its role in neuroinflammation is unknown. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules, and that haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to investigate the effects of TIA1 on neuroinflammation in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation including; reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress in hippocampal neurons and glia of wildtype and P301S transgenic mice expressing TIA1+/+, TIA1+/–, and TIA1–/– in both early (5 month) and advanced (9 month) disease states through biochemical, ultrastructural, and histological analyses. Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress.

Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Cyclooxygenase-2 (COX-2), with its main antifibrotic metabolite PGE2, is regarded as an antifibrotic gene. Repressed COX-2 expression and deficient PGE2 have been shown to contribute to the activation of lung fibroblasts and excessive deposition of collagen in pulmonary fibrosis. We have previously demonstrated that COX-2 expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) is epigenetically silenced and can be restored by epigenetic inhibitors. This study aimed to investigate whether COX-2 downregulation induced by the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in normal lung fibroblasts could be prevented by epigenetic inhibitors. We found that COX-2 protein expression and PGE2 production were markedly reduced by TGF-β1 and this was prevented by the pan-histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) and to a lesser extent by the DNA demethylating agent Decitabine (DAC), but not by the G9a histone methyltransferase (HMT) inhibitor BIX01294 or the EZH2 HMT inhibitor 3-deazaneplanocin A (DZNep). However, chromatin immunoprecipitation assay revealed that the effect of SAHA was unlikely mediated by histone modifications. Instead 3′-untranslated region (3′-UTR) luciferase reporter assay indicated the involvement of post-transcriptional mechanisms. This was supported by the downregulation by SAHA of the 3′-UTR mRNA binding protein TIA-1 (T-cell intracellular antigen-1), a negative regulator of COX-2 translation. Furthermore, TIA-1 knockdown by siRNA mimicked the effect of SAHA on COX-2 expression. These findings suggest SAHA can prevent TGF-β1-induced COX-2 repression in lung fibroblasts post-transcriptionally through a novel TIA-1-dependent mechanism and provide new insights into the mechanisms underlying its potential antifibrotic activity. AbbreviationsUnlabelled TableSAHAsuberanilohydroxamic acidTGF-β1transforming growth factor-β1COX-2cyclooxygenase-2TIA-1T-cell intracellular antigen-1PGE2prostaglandin E2IPFidiopathic pulmonary fibrosisDACDecitabineHMThistone methyltransferaseEZH2enhancer of zeste homolog 2DZNep3-deazaneplanocin A3′-UTR3′-untranslated regionα-SMAα-smooth muscle actinECMextracellular matrixCOL1collagen 1DNMTDNA methyltransferaseHAThistone acetyltransferaseHDAChistone deacetylaseH3K9me3histone H3 lysine 9 trimethylationAREAUUUA-rich elementHuRhuman antigen RELAV1ELAV-like RNA binding protein 1TTPTristetraprolinCUGBP2CUG triplet repeat, RNA binding protein 2F-NLfibroblast from non-fibrotic lungFCSfetal calf serum

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo.

SUMMARYEmerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S tau mice. Reducing TIA1 decreases the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibits the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles (NFTs). Despite the increase in NFTs, TIA1 reduction increases neuronal survival and rescues behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity, and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a paradigm in which dysfunction of the translational stress response leads to tau-mediated pathology.

Contrasting Pathology of the Stress Granule Proteins TIA-1 and G3BP in Tauopathies

Stress induces aggregation of RNA-binding proteins to form inclusions, termed stress granules (SGs). Recent evidence suggests that SG proteins also colocalize with neuropathological structures, but whether this occurs in Alzheimer's disease is unknown. We examined the relationship between SG proteins and neuropathology in brain tissue from P301L Tau transgenic mice, as well as in cases of Alzheimer's disease and FTDP-17. The pattern of SG pathology differs dramatically based on the RNA-binding protein examined. SGs positive for T-cell intracellular antigen-1 (TIA-1) or tristetraprolin (TTP) initially do not colocalize with tau pathology, but then merge with tau inclusions as disease severity increases. In contrast, G3BP (ras GAP-binding protein) identifies a novel type of molecular pathology that shows increasing accumulation in neurons with increasing disease severity, but often is not associated with classic markers of tau pathology. TIA-1 and TTP both bind phospho-tau, and TIA-1 overexpression induces formation of inclusions containing phospho-tau. These data suggest that SG formation might stimulate tau pathophysiology. Thus, study of RNA-binding proteins and SG biology highlights novel pathways interacting with the pathophysiology of AD, providing potentially new avenues for identifying diseased neurons and potentially novel mechanisms regulating tau biology.

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

Mutation of Mapped TIA-1/TIAR Binding Sites in the 3′ Terminal Stem-Loop of West Nile Virus Minus-Strand RNA in an Infectious Clone Negatively Affects Genomic RNA Amplification

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Cellular Protein TIA-1 Regulates the Expression of HBV Surface Antigen by Binding the HBV Posttranscriptional Regulatory Element

Objective: To identify T-cell intracellular antigen 1 (TIA-1) interacting with the posttranscriptional regulatory element (PRE) of hepatitis B virus (HBV), and reveal TIA-1 function on HBV gene expression. Methods: In order to demonstrate TIA-1 binding to PRE, electrophoretic mobility shift and RNA and protein pull-down assays were used. The pDM138-PRE reporter system, HepG2.2.15 cell line and HBs-PRE transient expression cells were adopted to identify TIA-1 function in the HBV life cycle. Results: TIA-1 could directly bind to PRE RNA. The functional binding region of TIA-1 was identified. Functional analysis indicated that TIA-1 could dose-dependently inhibit PRE function in pDM138-PRE system. Down-expression of HBs antigen induced by TIA-1 was detected both in the HepG2.2.15 cell line and in HBs-PRE transient expression cells. Conclusion: These data demonstrated that TIA-1 inhibits HBs expression by interacting with PRE.

The RNA-binding protein PTB exerts translational control on 3′-untranslated region of the mRNA for the ATP synthase β-subunit

We have recently reported that RNA-binding proteins TIA-1 (T-cell intracellular antigen-1), TIAR (TIA-1 related protein), and HuR (Hu antigen R) modulate the expression of the ATP synthase β-subunit mRNA (β-F1-ATPase mRNA) [J.M. Izquierdo, Control of the ATP synthase β subunit expression by RNA-binding proteins TIA-1, TIAR, and HuR, Biochem. Biophys. Res. Commun. 348 (2006) 703–711]. Here we found that PTB (Polypyrimidine Tract–Binding Protein) is a novel member of the ribonucleoprotein complex that interacts with the β-F1-ATPase mRNA through an adenosine/uridine (AU)-rich element located to the β-F1-ATPase 3′-untranslated region (β-3′-UTR). Co-expression of GFP from a reporter mRNA quimera containing human β-3′-UTR and recombinant PTB in HeLa cells increased the amount of GFP protein. Interestingly, this effect is not due to increased steady-state levels of GFP-β-3′-UTR mRNA. Taken together, these results suggest that PTB regulates post-transcriptional expression of the β-F1-ATPase mRNA at the translational level.

Control of the ATP synthase β subunit expression by RNA-binding proteins TIA-1, TIAR, and HuR

The β-subunit of the mitochondrial H +-ATP synthase (β-F1-ATPase) catalyzes the rate-limiting step of ATP formation in eukaryotic cells. Here, we examined the post-transcriptional regulation of human β-F1-ATPase mediated by the 3′-untranslated region of the mRNA (β-3′-UTR). Biochemical analysis revealed that the adenosine/uridine (AU)-rich element-binding proteins TIA-1 (T-cell intracellular antigen-1), TIAR (TIA-1-related protein), and HuR (Hu antigen R) interact with the β-F1-ATPase mRNA through an AU-rich sequence located to the 3′-UTR. Mouse embryonic fibroblasts (MEFs) knocked-out for TIA-1 or RNA interference (RNAi)-mediated knockdown of endogenous TIA-1, TIAR, or HuR in HeLa cells resulted in a decrease in β-F1-ATPase protein expression. The expression of GFP from a chimeric reporter containing human β-3′-UTR was also abolished in HeLa cells depleted of TIA-1, TIAR, or HuR. MEFs knocked-in for TIA-1 or the overexpression of RNAi-resistant TIA-1, TIAR, or HuR proteins in the RNAi-treated HeLa cells significantly restored the levels of the expression of both endogenous mouse β-F1-ATPase protein or recombinant GFP.

Identification and Functional Outcome of mRNAs Associated with RNA-Binding Protein TIA-1

The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-alpha) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-alpha and COX-2 mRNAs and in 3,019 additional UniGene transcripts (approximately 3% of the UniGene database), localizing preferentially to the 3' untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.