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.

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.

Splicing factor SRSF3 represses translation of p21cip1/waf1 mRNA.

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3'UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3'UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3.

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.

SNU-333 Cells as an Appropriate Cell Line for the Orthotopic Renal Cell Carcinoma Model.

Objective: To investigate a feasible candidate for an appropriate cell line for the orthotopic renal cell carcinoma (RCC) model. Methods: Normal human proximal tubule cells (HK-2) and RCC cells were used for MTT assay, Western blotting, sphere-forming assay, and orthotopic injection of BALB/c-nude mice. Immunohistochemistry was adopted in tissue arrays and orthotopic tumors. Results: Primary RCC cells showed resistance to a GPX4 inhibitor compared to HK-2 and to metastatic RCC cells, Caki-1. Caki-2 and SNU-333 cells showed resistance to ferroptosis via increased GPX4 and FTH1, respectively. RCC cells showed increased αSMA, in which Caki-2 and SNU-333 cells exhibited different epithelial-mesenchymal transition and cancer stem cell markers. Caki-1 and SNU-333 cells formed spheres in vitro and orthotopic tumor masses in vivo. The injected SNU-333 tumor only showed high intensities of CD10 and PAX8, markers of renal origin. Conclusion: SNU-333 cell line exhibited resistance via iron metabolism and stemness, and had tumor-initiating capacities in vitro and in vivo. These results suggest that among the cells tested, SNU-333 cells were the most promising for the establishment of an orthotopic RCC model for further researches.

Loss of MeCP2 causes subtle alteration in dendritic arborization of retinal ganglion cells.

Methyl-CpG-binding protein (MeCP2) is highly expressed in neurons. It plays an important role in the development of synapses and the formation of circuits in the central nervous system (CNS). Mutations in MECP2 cause neurodevelopmental disorders and mental retardation in humans. Therefore, it has become important to determine the distribution and function of MeCP2 in vivo. The retina consists of three nuclear cell layers and two layers of synapses; neurons in each layer are connected to form fine circuits necessary for visual signal transduction. Using immunohistochemical analysis, we found that MeCP2 was expressed in all nuclear cell layers, with differences in the levels of MeCP2 expression observed among the layers. To understand the structural defects in the retina due to the loss of MeCP2, we sought to elucidate the organization of the retinal structure in the Mecp2 knockout (KO) mouse. Overall, we found a normal retinal structure in Mecp2 KO mice. However, because Mecp2 mutations have a highly variable effect on neuronal architecture, we analyzed morphological changes in a subset of retinal ganglion cells of Mecp2 KO mice. In Thy1-GFP mice crossed with Mecp2 mutant mice, Sholl intersections analyses showed a subtle increase in number of intersections due to increased branching proximal to the soma in Mecp2 KO mice. Our results demonstrate that the expression of MeCP2 and the effects of Mecp2 mutations are highly specific to tissue and cell types.

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.

Stress-Induced Accumulation of HnRNP K into Stress Granules.

Stress granules (SGs) are cytoplasmic aggregates to reprogram gene expression in response to cellular stimulus. Here, we show that while SGs are being assembled in response to clotrimazole, an antifungal medication heterogeneous nuclear ribonucleoprotein (hnRNP) K, an RNA-binding protein that mediates translational silencing of mRNAs, is rapidly accumulated in SGs in U-2OS osteosarcoma cells. Forced expression of hnRNP K induces resistance to clotrimazole-induced apoptosis. Erk/MAPK is transiently activated in response to clotrimazole, and pharmacological suppression of the Erk/MAPK pathway sensitizes the cells to apoptosis. Inhibition of the Erk/MAPK pathway promotes the assembly of SGs. These results suggest that dynamic cytoplasmic formation of SGs and hnRNP K relocation to SGs may be defensive mechanisms against clotrimazole-induced apoptosis in U-2OS osteosarcoma cells.

The Role of Stress Granules in the Neuronal Differentiation of Stem Cells.

creativecommons.org/licenses/by-nc-sa/3.0/. Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NFM) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.

MeCP2 regulates gene expression through recognition of H3K27me3.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.

Translational downregulation of Twist1 expression by antiproliferative gene, B-cell translocation gene 2, in the triple negative breast cancer cells.

Twist1, a key transcription factor regulating epithelial-mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2/TIS21 expression. Based on our observation that forced expression of BTG2/TIS21 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2/TIS21-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2/TIS21-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2/TIS21 interacted with each other, which abrogated Twist1 activity. (2) BTG2/TIS21 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2/TIS21 maintained p-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-AA mutant expression and BTG2/TIS21 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2/TIS21-KO mouse than that in the wild type. (5) BTG2/TIS21-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2/TIS21 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2/TIS21 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2/TIS21-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2/TIS21 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2/TIS21 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2/TIS21 expression.

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.

Identifying the cellular location of brain cytoplasmic 200 RNA using an RNA-recognizing antibody.

Brain cytoplasmic 200 RNA (BC200 RNA) is a neuron-specific non-coding RNA, implicated in the inhibition of local synaptodendritic protein synthesis, and is highly expressed in some cancer cells. Although BC200 RNA has been shown to inhibit translation in vitro, the cellular location of this inhibition is unknown. In this study, we used a BC200 RNA-recognizing antibody to identify the cellular locations of BC200 RNA in HeLa cervical carcinoma cells. We observed punctate signals in both the cytoplasm and nucleus, and further discovered that BC200 RNA co-localized with the p-body decapping enzyme, DCP1A, and the heterogeneous nuclear ribonucleoprotein E2 (hnRNP E2). The latter is a known BC200 RNA-binding partner protein and a constituent of p-bodies. This suggests that BC200 RNA is localized to p-bodies via hnRNP E2. [BMB Reports 2017; 50(6): 318-322].

MicroRNA-1908-5p contributes to the oncogenic function of the splicing factor SRSF3.

Serine/arginine (SR)-rich proteins that contain RS domains and SR repeats have diverse cellular functions including transcription, polyadenylation, translation, and RNA export. The splicing factor SRSF3, also termed SRp20, is the smallest member of the SR protein family and is a known proto-oncogene. Although it is implicated in the malignant phenotypes of various cancer cells, the molecular mechanism underlying SRSF3-mediated cancer progression is still obscure. We investigated here the oncogenic functions of SRSF3 in osteosarcoma U2OS cells. Knockdown of SRSF3 inhibited proliferation, clonogenicity, and metastatic potential including migration and invasion. It also decreased the level of miR-1908 independent of its host gene FADS1. Although FADS1 was not associated with SRSF3-mediated malignant properties, overexpression of miR-1908-5p increased cell proliferation, migration, and invasion, suggesting that miR-1908-5p is responsible for the oncogenic functions of SRSF3. Knockdown of SRSF3 decreased the expression of miR-1908-5p by inhibiting transactivation of NF-κB. We observed that miR-1908-5p downregulated NF-κB inhibitor interacting Ras-like 2 (NKIRAS2), a negative regulator of the NF-κB pathway by directly binding to the 3'UTR of NKIRAS2 mRNA. Consistent with overexpression of miR-1908-5p, knockdown of NKIRAS2 diminished the expression level of IκB-ß and provoked translocation of NF-κB into the nucleus where it transcriptionally activates its target genes including miR-1908-5p expression, thus elevating the proliferation and metastatic potential. Taken together, our results demonstrate that SRSF3 confers the malignant characteristics on cancer cells via the SRSF3/miR-1908-5p/NKIRAS2 axis.

NEDDylation promotes stress granule assembly.

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Identification of Neuregulin-2 as a novel stress granule component.

Stress Granules (SGs) are microscopically visible, phase dense aggregates of translationally stalled messenger ribonucleoprotein (mRNP) complexes formed in response to distinct stress conditions. It is generally considered that SG formation is induced to protect cells from conditions of stress. The precise constituents of SGs and the mechanism through which SGs are dynamically regulated in response to stress are not completely understood. Hence, it is important to identify proteins which regulate SG assembly and disassembly. In the present study, we report Neuregulin-2 (NRG2) as a novel component of SGs; furthermore, depletion of NRG2 potently inhibits SG formation. We also demonstrate that NRG2 specifically localizes to SGs under various stress conditions. Knockdown of NRG2 has no effect on stress-induced polysome disassembly, suggesting that the component does not influence early step of SG formation. It was also observed that reduced expression of NRG2 led to marginal increase in cell survival under arsenite-induced stress. [BMB Reports 2016; 49(8): 449-454].

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.

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.

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.