The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression.

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau.

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

RNA partitioning into stress granules is based on the summation of multiple interactions.

Stress granules (SGs) are stress-induced RNA-protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA-protein, protein-protein, and RNA-RNA interactions with no single interaction dominating RNP recruitment into SGs.

RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of non-small cell lung cancer.

As the most common subtype of lung cancer, non-small cell lung cancer (NSCLC)is responsible for a large proportion of global cancer-caused deaths. The implication of long non-coding RNAs (lncRNAs) as tumor-suppressor or carcinogenic genes in NSCLC has been widely documented. Our study sought to investigate the performance of lncRNA RAMP2 antisense RNA1 (RAMP2-AS1) in NSCLC. GEPIA bioinformatics tool and RT-qPCR were applied for assessing the expression of RAMP2-AS1 and its neighboring gene receptor activity-modifying protein 2 (RAMP2) in NSCLC. Functional assays including CCK-8 assay, colony formation assay as well as caspase-3 activity analysis and Transwell invasion assays were applied for detecting the biological phenotypes of NSCLC cells. Interaction among RAMP2-AS1, RAMP2 and T-cell intracellular antigen 1cytotoxic granule associated RNA binding protein (TIA1) was evaluated by RNA immunoprecipitation and pulldown assays. We found that RAMP2-AS1 and RAMP2 were downregulated in NSCLC. Overexpression of RAMP2-AS1 hampered proliferation and invasion, whereas induced apoptosis of NSCLC cells. Mechanistically, RAMP2-AS1 interacted with TIA1 to stabilize the mRNA of RAMP2. In conclusion, we first uncovered that RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of NSCLC, providing new insight to improve the treatment efficacy of NSCLC.

Tandem RNA binding sites induce self-association of the stress granule marker protein TIA-1.

TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation.

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

The Multifunctional Faces of T-Cell Intracellular Antigen 1 in Health and Disease.

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is expressed in many tissues and in the vast majority of species, although it was first discovered as a component of human cytotoxic T lymphocytes. TIA1 has a dual localization in the nucleus and cytoplasm, where it plays an important role as a regulator of gene-expression flux. As a multifunctional master modulator, TIA1 controls biological processes relevant to the physiological functioning of the organism and the development and/or progression of several human pathologies. This review summarizes our current knowledge of the molecular aspects and cellular processes involving TIA1, with relevance for human pathophysiology.

Typical Stress Granule Proteins Interact with the 3' Untranslated Region of Enterovirus D68 To Inhibit Viral Replication.

Stress granules (SGs) are formed in the cytoplasm under environmental stress, including viral infection. Human enterovirus D68 (EV-D68) is a highly pathogenic virus which can cause serious respiratory and neurological diseases. At present, there is no effective drug or vaccine against EV-D68 infection, and the relationship between EV-D68 infection and SGs is poorly understood. This study revealed the biological function of SGs in EV-D68 infection. Our results suggest that EV-D68 infection induced the accumulation of SG marker proteins Ras GTPase-activated protein-binding protein 1 (G3BP1), T cell intracellular antigen 1 (TIA1), and human antigen R (HUR) in the cytoplasm of infected host cells during early infection but inhibited their accumulation during the late stage. Simultaneously, we revealed that EV-D68 infection induces HUR, TIA1, and G3BP1 colocalization, which marks the formation of typical SGs dependent on protein kinase R (PKR) and eIF2α phosphorylation. In addition, we found that TIA1, HUR, and G3BP1 were capable of targeting the 3' untranslated regions (UTRs) of EV-D68 RNA to inhibit viral replication. However, the formation of SGs in response to arsenite (Ars) gradually decreased as the infection progressed, and G3BP1 was cleaved in the late stage as a strategy to antagonize SGs. Our findings have important implications in understanding the mechanism of interaction between EV-D68 and the host while providing a potential target for the development of antiviral drugs.IMPORTANCE EV-D68 is a serious threat to human health, and there are currently no effective treatments or vaccines. SGs play an important role in cellular innate immunity as a target with antiviral effects. This manuscript describes the formation of SGs induced by EV-D68 early infection but inhibited during the late stage of infection. Moreover, TIA1, HUR, and G3BP1 can chelate a specific site of the 3' UTR of EV-D68 to inhibit viral replication, and this interaction is sequence and complex dependent. However, this inhibition can be antagonized by overexpression of the minireplicon. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets that can inhibit viral replication and limit the pathogenesis of EV-D68.

Identification of TIA1 mRNA targets during human neuronal development.

BACKGROUND: Neuronal development is a tightly controlled process involving multi-layered regulatory mechanisms. While transcriptional pathways regulating neurodevelopment are well characterized, post-transcriptional programs are still poorly understood. TIA1 is an RNA-binding protein that can regulate splicing, stability, or translation of target mRNAs, and has been shown to play critical roles in stress response and neurodevelopment. However, the identity of mRNAs regulated by TIA1 during neurodevelopment under unstressed conditions is still unknown. METHODS AND RESULTS: To identify the mRNAs targeted by TIA1 during the first stages of human neurodevelopment, we performed RNA immunoprecipitation-sequencing (RIP-seq) on human embryonic stem cells (hESCs) and derived neural progenitor cells (NPCs), and cortical neurons under unstressed conditions. While there was no change in TIA1 protein levels, the number of TIA1 targeted mRNAs decreased from pluripotent cells to neurons. We identified 2400, 845, and 330 TIA1 mRNA targets in hESCs, NPC, and neurons, respectively. The vast majority of mRNA targets in hESC were genes associated with neurodevelopment and included autism spectrum disorder-risk genes that were not bound in neurons. Additionally, we found that most TIA1 mRNA targets have reduced ribosomal engagement levels. CONCLUSION: Our results reveal TIA1 mRNA targets in hESCs and during human neurodevelopment, indicate that translation repression is a key process targeted by TIA1 binding and implicate TIA1 function in neuronal differentiation.

Differences in the immunosurveillance pattern associated with DNA mismatch repair status between right-sided and left-sided colorectal cancer.

Tumor location and immunity play important roles in the progression of colorectal cancer (CRC). This study aimed to investigate the differences in the immunosurveillance pattern between right- and left-sided CRC and analyze their association with clinicopathologic features, including mismatch repair (MMR) status. We included surgically resected stage II/III CRC cases and evaluated the immunohistochemical findings of HLA class I, HLA class II, programmed cell death-ligand 1 (PD-L1), PD-1, CTLA-4, CD3, CD4, CD8, TIA-1, T-bet, GATA3, RORγT, Foxp3, and CD163. A total of 117 patients were included in the analyses; of these, 30 and 87 had right- and left-sided cancer, respectively. Tumor immunity varied according to the tumor location in the overall cohort. Analysis of the tumors excluding those with DNA mismatch repair (MMR) deficiency also revealed that tumor immunity differed according to the tumor location. In right-sided colon cancer (CC), high expression of Foxp3 (P = .0055) and TIA-1 (P = .0396) were associated with significantly better disease-free survival (DFS). High CD8 (P = .0808) and CD3 (P = .0863) expression tended to have better DFS. Furthermore, in left-sided CRC, only high PD-L1 expression in the stroma (P = .0426) was associated with better DFS. In multivariate analysis, high Foxp3 expression in right-sided CC was an independent prognostic factor for DFS (hazard ratio, 7.6445; 95% confidence interval, 1.2091-150.35; P = .0284). In conclusion, the immunosurveillance pattern differs between right- and left-sided CRC, even after adjusting for MMR deficiency.

Zika Virus Subverts Stress Granules To Promote and Restrict Viral Gene Expression.

Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis.IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.

ALS-Linked Mutant SOD1 Associates with TIA-1 and Alters Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder caused by motor neuron loss. T-cell intracellular antigen-1 (TIA-1), a cytotoxic T lymphocyte granule-associated RNA binding protein, is a key component of stress granules. However, it remains uncertain whether ALS-causing superoxide dismutase-1 (SOD1) toxicity alters the dynamics of stress granules. Thus, through mouse and cell line models, and human cells and tissues, we showed the subcellular location of TIA-1 and its recruitment by stress granules following mutant SOD1-related stimuli. An overexpression of MTSOD1 resulted in increased TIA-1-positive cytoplasmic inclusions in the spinal cord tissue of SOD1G93A transgenic mouse and the SOD1G86S familial ALS patient. Moreover, we demonstrated the stages of ALS-like disease-dependent increase in TIA-1 in the spinal cord of transgenic mice. A similar increase of TIA-1 was found in the spinal cord of the SOD1G86S patient and induced pluripotent stem cell-derived neural stem cells from the SOD1G17S patient. By using immunoprecipitation assays in wild type (WT) human SOD1 (hSOD1) or mutant (MT) hSOD1-transfected motor neuronal cell lines and SOD1G93A transgenic mouse model, we observed that MTSOD1 interacts with TIA-1. In WT or MT hSOD1-transfected HEK293 and NSC-34 cells, the formation of TIA-1-positive stress granules was delayed in MTSOD1 by sodium arsenite treatment. These findings suggest that MTSOD1 could affect the dynamics of stress granules through the abnormal MTSOD1-TIA-1 interaction. Consequently, the resulting pathological TIA-1 may be involved in RNA metabolism found in ALS.

Hippo signaling promotes JNK-dependent cell migration.

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam-Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

Effects of annexin A7 inhibitor-ABO on the expression and distribution of long noncoding RNA-CERNA1 in vascular endothelial cells apoptosis.

More and more studies reported that diverse biological roles of long noncoding RNAs were usually dependent on their subcellular location. In our previous study, long noncoding RNA CERNA1 was identified both located in cytoplasm and nucleus of vascular endothelial cells (VECs). And CERNA1 in cytoplasm, which functioned as competitive endogenous RNA (ceRNA), alleviated the apoptosis of VECs. However, the function of CERNA1 in nucleus was still unclear. In this study, we found that nuclear CERNA1 positively regulated BCL2L10, which accelerated the serum and FGF-2 starvation-induced apoptosis of VECs, by enhancing the histone modification level of H3K9ac and H3K4me3 in BCL2L10 promoter region. Furthermore, due to the paradoxical function, we investigated the variation of CERNA1 subcellular location in VECs. The results showed that, as the change of apoptosis status, CERNA1 altered the cellular distribution in VECs. And the annexin A7 inhibitor, ABO (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine), not only increased the expression of CERNA1 by TIA-1, but also specifically improved its cytoplasm distribution proportion so as to inhibit the apoptosis of VECs. This evidence suggested that the subcellular location of CERNA1 played an important role in the VECs apoptosis and ABO might be a potential chemical molecule for therapy of VECs apoptosis related cardiovascular diseases.

KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation.

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi's sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.

TIA1 regulates the generation and response to toxic tau oligomers.

RNA binding proteins (RBPs) are strongly linked to the pathophysiology of motor neuron diseases. Recent studies show that RBPs, such as TIA1, also contribute to the pathophysiology of tauopathy. RBPs co-localize with tau pathology, and reduction of TIA1 protects against tau-mediated neurodegeneration. The mechanism through which TIA1 reduction protects against tauopathy, and whether TIA1 modulates the propagation of tau, are unknown. Previous studies indicate that the protective effect of TIA1 depletion correlates with both the reduction of oligomeric tau and the reduction of pathological TIA1 positive tau inclusions. In the current report, we used a novel tau propagation approach to test whether TIA1 is required for producing toxic tau oligomers and whether TIA1 reduction would provide protection against the spread of these oligomers. The approach used young PS19 P301S tau mice at an age at which neurodegeneration would normally not yet occur and seeding oligomeric or fibrillar tau by injection into hippocampal CA1 region. We find that propagation of exogenous tau oligomers (but not fibrils) across the brain drives neurodegeneration in this model. We demonstrate that TIA1 reduction essentially brackets the pathophysiology of tau, being required for the production of tau oligomers, as well as regulating the response of neurons to propagated toxic tau oligomers. These results indicate that RNA binding proteins modulate the pathophysiology of tau at multiple levels and provide insights into possible therapeutic approaches to reduce the spread of neurodegeneration in tauopathy.

Anaplastic large cell lymphoma with TP63 rearrangement: A dismal prognosis.

Anaplastic large cell lymphoma (ALCL) with TP63 rearrangement is a new entity and has the most dismal prognosis in all types of ALCL. This might be due to the resulting fusion protein having N-terminal truncated p63 with high oncogenic ability. Since this N-terminal domain has the function of tumor suppressor activity, the mechanism for high oncogenic capacity is thought to be the dominant negative function. Here, we report two ALCL cases with TP63 rearrangement that was each given too short a prognosis (Case 1 and 2: four and six months) in spite of intensive treatment. Immunohistochemically, p63 was highly expressed, and a sprit signal was detected using a TP63 break apart fluorescence in situ hybridization (FISH) in each case. Additionally, a poor prognostic marker of ALCL, all cytotoxic molecules (TIA-1, Granzyme B, and Perforin) were also expressed in almost all ALCL cells. Taken together, we suggest that not only the dominant negative function of N-truncated p63 but also the effect of cytotoxic molecules may influence the dismal prognosis of ALCL with TP63 rearrangement.

Deciphering the role of Zn2+-binding histidines from TIA-1 on the assembly and dynamics of stress granules.

T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes liquid-liquid phase separation as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+-mediated regulatory mechanism for protection against fibril formation in SGs.

The miR-30-5p/TIA-1 axis directs cellular senescence by regulating mitochondrial dynamics.

Senescent cells exhibit a diverse spectrum of changes in their morphology, proliferative capacity, senescence-associated secretory phenotype (SASP) production, and mitochondrial homeostasis. These cells often manifest with elongated mitochondria, a hallmark of cellular senescence. However, the precise regulatory mechanisms orchestrating this phenomenon remain predominantly unexplored. In this study, we provide compelling evidence for decreases in TIA-1, a pivotal regulator of mitochondrial dynamics, in models of both replicative senescence and ionizing radiation (IR)-induced senescence. The downregulation of TIA-1 was determined to trigger mitochondrial elongation and enhance the expression of senescence-associated ß-galactosidase, a marker of cellular senescence, in human foreskin fibroblast HS27 cells and human keratinocyte HaCaT cells. Conversely, the overexpression of TIA-1 mitigated IR-induced cellular senescence. Notably, we identified the miR-30-5p family as a novel factor regulating TIA-1 expression. Augmented expression of the miR-30-5p family was responsible for driving mitochondrial elongation and promoting cellular senescence in response to IR. Taken together, our findings underscore the significance of the miR-30-5p/TIA-1 axis in governing mitochondrial dynamics and cellular senescence.

Chandipura Virus Forms Cytoplasmic Inclusion Bodies through Phase Separation and Proviral Association of Cellular Protein Kinase R and Stress Granule Protein TIA-1.

Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.