Dihydrocapsaicin induces translational repression and stress granule through HRI-eIF2α phosphorylation axis.

Stress granules (SGs) are cytoplasmic biomolecular condensates that are formed against a variety of stress conditions when translation initiation is perturbed. SGs form through the weak protein-protein, protein-RNA, and RNA-RNA interactions, as well as through the intrinsically disordered domains and post-translation modifications within RNA binding proteins (RBPs). SGs are known to contribute to cell survivability by minimizing the stress-induced damage to the cells by delaying the activation of apoptosis. Here, we find that dihydrocapsaicin (DHC), an analogue of capsaicin, is a SG inducer that promotes polysome disassembly and reduces global protein translation via phosphorylation of eIF2α. DHC-mediated SG assembly is controlled by the phosphorylation of eIF2α at serine 51 position and is controlled by all four eIF2α stress kinases (i.e., HRI, PKR, PERK, and GCN2) with HRI showing maximal effect. We demonstrate that DHC is a bonafide compound that induces SG assembly, disassembles polysome, phosphorylates eIF2α in an HRI dependent manner, and thereby arrest global translation. Together, our results suggest that DHC is a novel SG inducer and an alternate to sodium arsenite to study SG dynamics.

Oxidative stress-induced aberrant G9a activation disturbs RE-1-containing neuron-specific genes expression, leading to degeneration in human SH-SY5Y neuroblastoma cells.

Oxidative stress-induced neurodegeneration is one of several etiologies underlying neurodegenerative disease. In the present study, we investigated the functional role of histone methyltransferase G9a in oxidative stress-induced degeneration in human SH-SY5Y neuroblastoma cells. Cell viability significantly decreased on H2O2 treatment; however, treatment with the G9a inhibitor BIX01294 partially attenuated this effect. The expression of neuron-specific genes also decreased in H2O2- treated cells; however, it recovered on G9a inhibition. H2O2-treated cells showed high levels of H3K9me2 (histone H3 demethylated at the lysine 9 residue), which is produced by G9a activation; BIX01294 treatment reduced aberrant activation of G9a. H3K9me2 occupancy of the RE-1 site in neuron-specific genes was significantly increased in H2O2-treated cells, whereas it was decreased in BIX01294-treated cells. The differentiation of H2O2-treated cells also recovered on G9a inhibition by BIX01294. Consistent results were observed when used another G9a inhibitor UCN0321. These results demonstrate that oxidative stress induces aberrant activation of G9a, which disturbs the expression of neuron-specific genes and progressively mediates neuronal cell death. Moreover, a G9a inhibitor can lessen aberrant G9a activity and prevent neuronal damage. G9a inhibition may therefore contribute to the prevention of oxidative stress-induced neurodegeneration.

SRSF3-regulated miR-132/212 controls cell migration and invasion by targeting YAP1.

Although SRSF3 (Serine/arginine-rich splicing factor 3) plays a significant role in various biological processes, many of its functions still remain unclear. More particularly, little is known about SRSF3's involvement in the regulation of miRNA. In this report, we found that invasive and migratory abilities were inhibited in SRSF3-silenced U2OS and HeLa cells. We also found that a knockdown of SRSF3 results in a decreased expression level of REST (RE1-silencing transcription factor). The silencing of REST increased the expression of primary miR-132/212 as well as their mature forms. In particular, miR-132-3p and miR-212-3p possess an identical seed sequences and a common target gene. Overexpression of miR-132-3p and miR-212-3p suppressed the expression of YAP1 (Yes-associated protein 1) by directly binding to the 3՚UTR of its mRNA. CCND1 (Cyclin D1), which acts downstream of YAP1, was downregulated in both miR-132-3p and miR-212-3p-overexpressed cells, in correlation with diminished YAP1 levels. Taken together, our results reveal that SRSF3 controls the expression of the miR-132/212 cluster through regulating REST expression, and that the REST-elicited alteration of miRNA expression is implicated in enabling the migratory and invasive abilities of cancer cells.

Splicing inhibition of U2AF65 leads to alternative exon skipping.

U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or ß-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.

Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets.

Ischemic brain extract increases SDF-1 expression in astrocytes through the CXCR2/miR-223/miR-27b pathway.

Ischemic cerebral stroke is one of the leading global causes of mortality and morbidity. Ischemic preconditioning (IPC) refers to a sublethal ischemia and resulting in tolerance to subsequent severe ischemic injury. Although several pathways are reportedly involved in IPC-mediated neuroprotection, the functional role of astrocytes is not fully understood. Stromal cell-derived factor-1 (SDF-1), a CXC chemokine produced mainly in astrocytes, is a ligand for chemokine receptor CXCR4. SDF-1 is reported to play a critical role in neuroprotection after stroke by mediating the migration of neuronal progenitor cells. We hypothesized that stimuli derived from ischemic brain were involved in the protective effects of IPC. To investigate this hypothesis, the mechanism in which ischemic brain extract (IBE) induced SDF-1 expression was investigated in C6 astrocytoma cells. IBE treatment of C6 cells increased SDF-1 expression compared to that in untreated or normal brain extract (NBE)-treated cells by downregulating SDF-1 targeting miRNA, miR-27b. MiR-223 was inversely upregulated in IBE-treated cells; overexpression of miR-223 decreased the expression of miR-27b by suppressing IKKα expression. Analysis of cytokine array data revealed an IBE associated enhanced expression of CINC-1 (CXCL1) and LIX1 (CXCL5). Knockdown or inhibition of their receptor, CXCR2, abolished IBE-mediated increased expression of SDF-1. These results were confirmed in primary cultured astrocytes. Taken together, the data demonstrate that IBE-elicited signals increase SDF-1 expression through the CXCR2/miR-223/miR-27b pathway in C6 astrocytoma cells and primary astrocytes, supporting the view that increased expression of SDF-1 by ischemic insults is a possible mechanism underlying therapeutic application of IPC.

Regulation of cellular RNA nano-particle assembly by splicing factor SRp20.

Cellular RNA nano-particles (RNA granules) such as stress granule (SG) and P-body (PB) are translationally silenced mRNA-protein complexes. Previously, a genome-wide loss-of-function screen using oligomeric siRNAs targeting potential drug target genes was performed to identify genes that are involved in SG and PB assembly. SRp20 (SRSF3), a splicing regulator, was identified as a potential regulator for the RNA granule assembly. Here, we show that SRp20 is a bona-fide RNA granule component using antibody against SRp20 as well as Flag-tagged SRp20 through immunofluorescence microscopy. More importantly, upon knockdown of SRp20 using siRNA, RNA granule formation was potently disrupted indicating that SRp20 is one of the major structural components of SGs and PBs. Interestingly, polysome profiling analyses displayed that SRp20 is distributed in all ribosomal fractions suggesting a potential role of SRp20 as a post-transcriptional mRNA regulator. These results broaden the functional role of SRp20 from the nuclear RNA processing events to the cytoplasmic post-transcriptional mRNA regulatory events through RNA granules that are critical for the regulation of gene expression.

METTL3 stabilization by PIN1 promotes breast tumorigenesis via enhanced m6A-dependent translation.

Methyltransferase-like 3 (METTL3) is the catalytic subunit of the N6-adenosine methyltransferase complex responsible for N6-methyladenosine (m6A) modification of mRNA in mammalian cells. Although METTL3 expression is increased in several cancers, the regulatory mechanisms are unclear. We explored the regulatory roles of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) in METTL3 stability and m6A modification of mRNA. PIN1 interacted with METTL3 and prevented its ubiquitin-dependent proteasomal and lysosomal degradation. It stabilized METTL3, which increased the m6A modification of transcriptional coactivator with PDZ-binding motif (TAZ) and epidermal growth factor receptor (EGFR) mRNA, resulting in their efficient translation. PIN1 knockout altered the distribution of TAZ and EGFR mRNA from polysomes into monosomes. Inhibition of MEK1/2 kinases and PIN1 destabilized METTL3, which impeded breast cancer cell proliferation and induced cell cycle arrest at the G0/G1 phases. METTL3 knockout reduced PIN1 overexpression-induced colony formation in MCF7 cells and enhanced tumor growth in 4T1 cells in an orthotopic mouse model. In clinical settings, METTL3 expression significantly increased with tumor progression and was positively correlated with PIN1 expression in breast cancer tissues. Thus, PIN1 plays a regulatory role in mRNA translation, and the PIN1/METTL3 axis may be an alternative therapeutic target in breast cancer.

Targeting the multidrug and toxin extrusion 1 gene (SLC47A1) sensitizes glioma stem cells to temozolomide.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, with an extremely poor prognosis due to resistance to standard-of-care treatments. Strong evidence suggests that the small population of glioma stem cells (GSCs) contributes to the aggressiveness of GBM. One of the mechanisms that promote GSC progression is the dysregulation of membrane transporters, which mediate the influx and efflux of substances to maintain cellular homeostasis. Here, we investigated the role of multidrug and toxin extrusion transporter gene SLC47A1 in GSCs. Results show that SLC47A1 is highly expressed in GSCs compared to non-stem cell glioma cells, and non-tumor cells. Additionally, in-silico analysis of public datasets showed that high SLC47A1 expression is linked to malignancy and a poor prognosis in glioma patients. Further, SLC47A1 expression is correlated with important biological processes and signaling pathways that support tumor growth. Meanwhile, silencing SLC47A1 by short-hairpin RNA (shRNA) influenced cell viability and self-renewal activity in GSCs. Interestingly, SLC47A1 shRNA knockdown or pharmacological inhibition potentiates the effect of temozolomide (TMZ) in GSC cells. The findings suggest that SLC47A1 could serve as a useful therapeutic target for gliomas.

Proteomic analysis of the copper ion-induced stress response in a human embryonic carcinoma cell line.

Excessive exposure to copper, a redox-active metal, generates free radicals, which can cause cellular damage. In this study, we aim to identify the proteins that are up- or downregulated by copper exposure in human embryonic carcinoma (NCCIT) cells and to understand the mechanisms that play a role in the copper-induced stress response. After exposure to copper ions, the cells showed upregulated levels of 78 kDa glucose-regulated protein, fibrillin 1, CWC22 spliceosome-associated protein (KIAA1604), heat shock protein (HSP) 60, and HSP70, while the tumor necrosis factor receptor-associated factor 6, vimentin, 14-3-3 protein zeta, and RAC-beta (AKT2) serine/threonine protein kinase were downregulated. The GeneGo Process Networks of the proteins upregulated by copper ions were analyzed, and the 3 highest-scoring networks from the proteins upregulated by copper ions are presented here. In particular, the increased level of HSP70 in response to copper ions occurred in a dose-dependent manner, indicating that HSP70 could be a potential biomarker for copper toxicity in mammalian cells.