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Sublethal cell damage can trigger a complex adaptive program known as senescence, characterized by growth arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP). As senescent cells accumulating in aging organs are linked to many age-associated diseases, senotherapeutic strategies are actively sought to eliminate them. Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP-TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, Verteporfin (VPF), selectively triggered apoptotic cell death and derepressed DDIT4, in turn inhibiting mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, causing ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased senescent cell numbers in the organs of old mice and mice exhibiting doxorubicin-induced senescence. We present a novel senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

Circular RNAs are abundant, covalently closed transcripts that arise in cells through back-splicing and display distinct expression patterns across cells and developmental stages. While their functions are largely unknown, their intrinsic stability has made them valuable biomarkers in many diseases. Here, we set out to examine circRNA patterns in amyotrophic lateral sclerosis (ALS). By RNA-sequencing analysis, we first identified circRNAs and linear RNAs that were differentially abundant in skeletal muscle biopsies from ALS compared to normal individuals. By RT-qPCR analysis, we confirmed that 8 circRNAs were significantly elevated and 10 were significantly reduced in ALS, while the linear mRNA counterparts, arising from shared precursor RNAs, generally did not change. Several of these circRNAs were also differentially abundant in motor neurons derived from human induced pluripotent stem cells (iPSCs) bearing ALS mutations, and across different disease stages in skeletal muscle from a mouse model of ALS (SOD1

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.

Long noncoding (lnc)RNAs potently regulate gene expression programs in physiology and disease. Here, we describe a key function for lncRNA OIP5-AS1 in myogenesis, the process whereby myoblasts differentiate into myotubes during muscle development and muscle regeneration after injury. In human myoblasts, OIP5-AS1 levels increased robustly early in myogenesis, and its loss attenuated myogenic differentiation and potently reduced the levels of the myogenic transcription factor MEF2C. This effect relied upon the partial complementarity of OIP5-AS1 with MEF2C mRNA and the presence of HuR, an RNA-binding protein (RBP) with affinity for both transcripts. Remarkably, HuR binding to MEF2C mRNA, which stabilized MEF2C mRNA and increased MEF2C abundance, was lost after OIP5-AS1 silencing, suggesting that OIP5-AS1 might serve as a scaffold to enhance HuR binding to MEF2C mRNA, in turn increasing MEF2C production. These results highlight a mechanism whereby a lncRNA promotes myogenesis by enhancing the interaction of an RBP and a myogenic mRNA.

In older persons, senescent vascular smooth muscle cells (VSMCs) express increased levels of senescence-associated secretory phenotype (SASP) factors such as cytokines, chemokines, adhesion molecules, matrix-remodeling enzymes, and angiogenic factors. The SASP contributes to destabilizing the plaque cap, suggesting that VSMC senescence promotes disease development. As a result of their prevalence in many diseases, major efforts are underway to develop therapies directed at senescent cells (senotherapies). Dipeptidyl Peptidase 4 (DPP4), a serine protease, was elevated on the plasma membrane of senescent fibroblasts. While the effect of DPP4 on VSMCs is not well understood, DPP4 inhibitors such as Vildagliptin are clinically used to treat diabetes, and in animal models, gliptins reduced atherosclerosis and inflammation independently of DPP4’s canonical role in glucose metabolism. We hypothesized that DPP4 inhibitors may reduce the progression and burden of atherosclerosis by preventing DPP4 enzymatic function on senescent VSMCs. Towards this end, we discovered that DPP4 protein levels and enzymatic activity increased in senescent human VSMCs (hVSMCs) senescence. We also found elevated DPP4 in human atherosclerotic plaques, and high levels of DPP4 colocalized with the senescence marker p16 in murine atherosclerosis. Strikingly, silencing DPP4 in senescent hVSMCs increased cell death and caspase activity. Proteomic analysis of conditioned media from senescent hVSMCs treated with DPP4 inhibitors revealed a reduction in numerous complement and coagulation factors, indicating that DPP4 partially governs hemostasis and the proteolytic events that promote thrombotic signaling. We propose that DPP4 inhibition suppresses the pro-coagulation/complement phenotype of senescent hVSMCs to disrupt their function and improve vascular disease progression.

Changes in the proteome of different human tissues with advancing age are poorly characterized. Here, we studied the proteins present in primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Proteins were extracted from lysed fibroblasts and subjected to liquid chromatography-mass spectrometry analysis, and the expression levels of 9341 proteins were analyzed using linear regression models. We identified key pathways associated with skin fibroblast aging, including autophagy, scavenging of reactive oxygen species (ROS), ribosome biogenesis, DNA replication, and DNA repair. Changes in these prominent pathways were corroborated using molecular and cell culture approaches. Our study establishes a framework of the global proteome governing skin fibroblast aging and points to possible biomarkers and therapeutic targets.

Although some long noncoding (lnc)RNAs are known since the 1950s, the past 25 years have uncovered myriad lncRNAs with diverse sequences, structures, and functions. The advent of high-throughput and sensitive technologies has further uncovered the vast heterogeneity of lncRNA-interacting molecules and patterns of expressed lncRNAs. We propose a unifying functional theme for the expansive family of lncRNAs. At an elementary level, the genomic program of gene expression is elicited via canonical transcription and post-transcriptional mRNA assembly, turnover, and translation. Building upon this regulation, an epigenomic program refines the basic genomic control by modifying chromatin architecture as well as DNA and RNA chemistry. Superimposed over the genomic and epigenomic programs, lncRNAs create an additional regulatory dimension: by interacting with the proteins and nucleic acids that regulate gene expression in the nucleus and cytoplasm, lncRNAs help establish robust, nimble, and specific transcriptional and post-transcriptional control. We describe our present understanding of lncRNA-coordinated control of protein programs and cell fate, and discuss challenges and opportunities as we embark on the next 25 years of lncRNA discovery.

Malignant characteristics of cancers, represented by rapid cell proliferation and high metastatic potential, are a major cause of high cancer-related mortality. As a multifunctional RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK) is closely associated with cancer progression in various types of cancers. In this study, we sought to identify hnRNPK-regulated long intergenic non-coding RNAs (lincRNAs) that play a critical role in the regulation of cancer malignancy. We found that hnRNPK controlled malignant phenotypes including invasiveness, proliferation, and clonogenicity. RNA sequencing and functional studies revealed that LINC00263, a novel target of hnRNPK, is involved in the oncogenic functions of hnRNPK. Knockdown of LINC00263 mitigated the malignant capabilities. Conversely, increased malignant phenotypes were observed in LINC00263-overexpressing cells. Since LINC00263 was mainly localized in the cytosol and highly enriched in Argonaute 2-immunoprecipitation (Ago2-IP), we hypothesized that LINC00263 acts as a competitive endogenous RNA (ceRNA), and thus sought to identify LINC00263-associated microRNAs. Using small RNA sequencing followed by antisense oligonucleotide pull-down, miR-147a was selected for further study. We found that miR-147a negatively regulates LINC00263 via direct interaction, thus suppressing malignant capabilities. Moreover, knockdown of hnRNPK and LINC00263 upregulated miR-147a, indicating that LINC00263 serves as a ceRNA for miR-147a. By analyzing RNA sequencing data and miRNA target prediction, calpain 2 (CAPN2) was identified as a putative target of miR-147a. Ago2-IP and luciferase reporter assay revealed that miR-147a suppressed CAPN2 expression by directly binding to the 3′UTR of CAPN2 mRNA. In addition, we found that the weakened malignant capabilities following knockdown of hnRNPK or LINC00263 were restored by miR-147a inhibition or CAPN2 overexpression. Furthermore, our findings were validated in various other types of cancer cells including lung cancer, colorectal cancer, neuroblastoma, and melanoma. Collectively, we demonstrate that hnRNPK-regulated LINC00263 plays an important role in cancer malignancy by acting as a miR-147a decoy and thus upregulating CAPN2.

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45 ( + ) /CD11b ( + ) macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.77974

Recent technological advances in RNA sequencing and analysis have allowed an increasingly thorough investigation of a previously unexplored class of transcripts, circular (circ)RNAs. Accumulating evidence suggests that circRNAs have unique functions which often rely on their association with microRNAs and RNA-binding proteins. Through these interactions, circRNAs have been implicated in major cellular processes and hence in the pathophysiology of a range of diseases. Here, we provide guidelines to consider when developing studies on circRNAs, including detecting and selecting the circRNAs, identifying their binding partners and sites of interaction, modulating circRNA levels, assessing copy numbers and stoichiometry, and addressing other points unique to circRNA analysis. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2 Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G1-S progression upon silencing circ-MDM2 Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1 mM. In contrast, low and moderate doses of DEM (10–100 µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100 µM DEM, but not 1 mM DEM, whereas only 1 mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms., ISSN:2213-2317, Redox Biology, 11

// Dimitrios Tsitsipatis 1, 6 , Ashok Kumar Jayavelu 1, 7 , Jorg P. Muller 1 , Reinhard Bauer 1 , Dirk Schmidt-Arras 2 , Siavosh Mahboobi 3 , Tina M. Schnoder 4, 5 , Florian Heidel 4, 5 , Frank-D. Bohmer 1 1 Institute of Molecular Cell Biology, CMB, Jena University Hospital, Jena, Germany 2 Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany 3 Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Regensburg, Germany 4 Innere Medizin II, Hamatologie und Onkologie, Universitatsklinikum Jena, Jena, Germany 5 Leibniz Institute on Aging, Fritz-Lipmann-Institute, Jena, Germany 6 Current address: Institute of Nutrition, Department of Nutrigenomics, Friedrich-Schiller-University, Jena, Germany 7 Current address: Max-Planck Institute of Biochemistry, Department of Proteomics and Signal Transduction, Martinsried, Germany Correspondence to: Frank-D. Bohmer, email: boehmer@med.uni-jena.de Keywords: acute myeloid leukemia, FLT3ITD, tunicamycin, selective cytotoxicity Received: May 11, 2016 Accepted: February 16, 2017 Published: February 28, 2017 ABSTRACT Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein ( CHOP ). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2. Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo. Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G(1)-S progression upon silencing circ-MDM2. Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

Noncoding RNAs have substantial effects in host–virus interactions. Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs which can decoy other RNAs or RNA-binding proteins to inhibit their functions. The role of circRNAs is largely unknown in the context of Kaposi’s sarcoma herpesvirus (KSHV). We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Transcriptome analysis of KSHV-infected primary endothelial cells and a B cell line identified human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with divergent PCR primers and RNase R treatment of specific circRNAs. Ectopic expression of hsa_circ_0001400, a circRNA induced by infection, suppressed expression of key viral latent gene LANA and lytic gene RTA in KSHV de novo infections. Since human herpesviruses express noncoding RNAs like microRNAs, we searched for viral circRNAs encoded in the KSHV genome. We performed circRNA-Seq analysis with RNase R-treated, circRNA-enriched RNA from KSHV-infected cells. We identified multiple circRNAs encoded by the KSHV genome that are expressed in KSHV-infected endothelial cells and primary effusion lymphoma (PEL) cells. The KSHV circRNAs are located within ORFs of viral lytic genes, are up-regulated upon the induction of the lytic cycle, and alter cell growth. Viral circRNAs were also detected in lymph nodes from patients of KSHV-driven diseases such as PEL, Kaposi’s sarcoma, and multicentric Castleman’s disease. We revealed new host–virus interactions of circRNAs: human antiviral circRNAs are activated in response to KSHV infection, and viral circRNA expression is induced in the lytic phase of infection.

Cellular senescence, a developmental program central to normal aging and aging pathologies, is robustly regulated at the post-transcriptional level. This regulation involves the interaction of RNA-binding proteins and noncoding RNAs with senescence-associated messenger RNAs (mRNAs). There is increasing evidence that these associations are modulated by chemical modifications of specific mRNA nucleotides which can enhance or reduce the binding of regulatory factors. Recent technological advances in mass spectrometry, next-generation sequencing, and genome mapping have improved markedly the detection of mRNA modifications. Given the rising interest in the epitranscriptomic control of gene expression in aging, we discuss our incipient understanding of the chemical mRNA modifications, specifically m6 A and m5 C, that influence cellular senescence. This article is categorized under: RNA Export and Localization > RNA Localization RNA Processing > RNA Editing and Modification.

Diethyl maleate (DEM), a thiol-reactive α,β-unsaturated carbonyl compound, depletes glutathione (GSH) in exposed cells and was previously shown by us to elicit a stress response in Caenorhabditis elegans that, at lower concentrations, results in enhanced stress resistance and longer lifespan. This hormetic response was mediated through both the Nrf2 ortholog, SKN-1, and the forkhead box O (FOXO) family transcription factor DAF-16. As FOXO signaling is evolutionarily conserved, we analyzed here the effects of DEM exposure on FOXO in cultured human cells (HepG2, HEK293). DEM elicited nuclear accumulation of GFP-coupled wild-type human FOXO1, as well as of a cysteine-deficient FOXO1 mutant. Despite the nuclear accumulation of FOXO1, neither FOXO1 DNA binding nor FOXO target gene expression were stimulated, suggesting that DEM causes nuclear accumulation but not activation of FOXO1. FOXO1 nuclear exclusion elicited by insulin or xenobiotics such as arsenite or copper ions was attenuated by DEM, suggesting that DEM interfered with nuclear export. In addition, insulin-induced FOXO1 phosphorylation at Thr-24, which is associated with FOXO1 nuclear exclusion, was attenuated upon exposure to DEM. Different from FOXO-dependent expression of genes, Nrf2 target gene mRNAs were elevated upon exposure to DEM. These data suggest that, different from C. elegans, DEM elicits opposing effects on the two stress-responsive transcription factors, Nrf2 and FOXO1, in cultured human cells. Keywords: FOXO, DAF-16, Glutathione, Stress response, Nrf-2, Thiols, Nuclear export

Hepatic production and release of metabolites, nutrients and micronutrient transporters is tightly regulated at the level of gene expression. In this regard, transcription factor FOXO1 modulates the expression of genes such as G6PC and SELENOP, encoding the catalytic subunit of glucose 6-phosphatase and the plasma selenium transporter selenoprotein P, respectively. Here, we analyzed the role of cysteine residues in FOXO1 in controlling its activity with respect to regulation of G6PC and SELENOP in HepG2 human hepatoma cells. None of the seven FOXO1 cysteines affected FOXO1 binding to DNA or its basal subcellular distribution. Whereas overexpression of wildtype FOXO1 caused a strong induction of both G6PC and SELENOP promoter activities and mRNA levels, the induction was lowered by approx. 50% if cysteine-deficient FOXO1 was overexpressed instead. Only the most C-terminal of the seven FOXO1 cysteines, Cys612, was required and sufficient to ensure full FOXO1 transactivation activity. Coexpression of FOXO1 coregulators, CBP or PGC1α, had a strong synergistic effect in stimulating G6PC promoter activity and expression, fully relying on the presence of FOXO1 Cys612. Similarly, a synergistic effect of FOXO1 and CBP was observed for SELENOP. In contrast, stimulation of SELENOP by PGC1α was independent of FOXO1-Cys612, due to the close proximity of a hepatocyte nuclear factor-4α binding site to the FOXO1 binding site within the SELENOP promoter, as demonstrated using mutant SELENOP promoter constructs. In summary, full basal FOXO1 transactivation activity relies on Cys612, which mediates synergistic effects of coregulators, CBP or PGC1α, on FOXO1 transcriptional activity. The extent of Cys612 contribution depends on the promoter context of FOXO1 target genes.

Background The ubiquitously expressed forkhead box, class O (FoxO) transcription factors act as signaling integrators in extensive transcriptional networks, ensuring maintenance of cell and tissue homeostasis over time and in response to environmental challenges. Proteins whose biosynthesis is controlled through FoxOs fulfil key functions in antioxidant defense, metabolism, cell cycle regulation and apoptosis. Scope of review All four mammalian FoxO isoforms (FoxO1, FoxO3, FoxO4 and FoxO6) are expressed in skin but functions have been specified only for FoxO1 and FoxO3. This review provides an overview on the roles of FoxO1 and FoxO3 in the major types of skin cells: fibroblasts, keratinocytes and melanocytes. Major conclusions As expected because of their target genes, FoxOs are involved in counter-acting oxidative stress and in decisions on cell fate regarding apoptosis or senescence. However, their role in skin surpasses these rather obvious tasks: FoxO1 is part of signaling axes related to the control of epidermal morphogenesis and the pathogenesis of acne. FoxO3 dampens the biosynthesis of melanin in melanocytes; on the other hand, FoxO3 suppression in melanoma is associated with impaired apoptosis and increased metastatic potential of melanoma cells. Upon skin injury, a well-balanced and -timed up-regulation of FoxOs appears to support the healing process through affecting proliferation, migration and apoptosis of keratinocytes, fibroblasts and other cells accumulating at the wounded site. General significance FoxO1 and FoxO3 are discussed as homeostatic factors that influence morphogenesis, maintenance and repair processes in skin as well as the pathogenesis of disorders such as acne and skin cancer.

FOXO transcription factors regulate several aspects of the cellular stress response and antioxidant defence. It has been known for some time that FOXO cysteine residues are essential for oxidative stress-induced interactions with coregulators such as CBP/p300, which reversibly interacts with FOXO4 upon exposure to H2O2. Here, we investigated whether cysteine residues are essential for FOXO1 activity under basal conditions. To this end, we generated epitope-tagged cysteine-deficient mutants of human FOXO1 (all 7 cysteines were replaced by serines). Cysteine deficiency did not impair FOXO1 DNA binding, as determined using EMSAs, nor was nucleocytoplasmic distribution and shuttling affected in HEK293 or HepG2 cells overexpressing WT or Cys-deficient FOXO1. In contrast, stimulation of FOXO-responsive promoter constructs by overexpression of FOXO1 was far less prominent in cells overexpressing Cys-deficient FOXO1 as compared to those transfected with WT-FOXO1. Similarly, FOXO1 overexpression-induced increases in FOXO target gene mRNA levels was impaired in Cys-deficient FOXO1 relative to WT-FOXO1. A series of single-Cys mutation experiments led us to conclude that, of all 7 cysteines, only C612 is required for full FOXO1 transactivation activity under basal, non-stressed conditions. The exact role of C612 in controlling FOXO1 activity remains to be established.

Cysteine-S-glutathionylation is a reversible post-translational modification occurring under oxidative stress. Here, we tested how transcription factor FoxO1a activity is affected by the absence of its cysteine residues and whether glutathionylation of FoxO1a might occur. A V5-tagged cysteine-deficient mutant of human FoxO1a (all 7 cysteines were replaced by serines) was generated by site directed mutagenesis, followed by transfection of HepG2 human hepatoma cells or HEK293 human embryonic kidney cells to overexpress either wild type (WT) or Cys-deficient V5-FoxO1a. Neither insulin-induced phosphorylation of FoxO1a nor its nucleocytoplasmic shuttling was attenuated in cysteine-deficient FoxO1a. Moreover, no alteration of basal FoxO1a DNA binding activity (EMSA) was noticed in the Cys-deficient version versus WT under normal culture conditions. However, exposure to diamide, a thiol-oxidizing agent, revealed that, while FoxO1a-DNA interaction was attenuated in the WT form, this oxidant-induced attenuation was less prominent in the cysteine-deficient mutant. Furthermore, transactivation of a FoxO-responsive element-driven reporter gene was less prominent in cells overexpressing Cys-deficient FoxO1a than in those transfected with WT FoxO1a. Immunoprecipitation of glutathionylated proteins in cells exposed to diamide co-precipitated WT-FoxO1a more efficiently than Cys-deficient FoxO1a. Moreover, Western blotting analyses (reducing/non-reducing) of diamide-exposed cells overexpressing WT or mutant FoxO1a suggest that oxidant-induced FoxO1a interaction with cofactors is attenuated in the Cys-deficient mutant. In summary, these data suggest that cysteine residues in FoxO1a, while not affecting insulin-induced phosphorylation and nucleocytoplasmic shuttling, are important mediators of FoxO1a/DNA interaction under conditions of oxidative stress. Moreover, these data point to glutathionylation of FoxO1a and/or yet to be identified cofactors under exposure to oxidants, suggesting a regulation of FoxO activity through S-glutathionylation.

Diethyl maleate (DEM) is a thiol-depleting agent frequently employed in cell culture analyses. Here, we investigated the effect of DEM exposure on insulin signaling at the level of FoxO transcription factor activity and its potential consequences for stress resistance and life span. Exposure of HepG2 human hepatoma cells to subcytotoxic concentrations of DEM resulted in nuclear accumulation of overexpressed EGFP-tagged FoxO1a. DEM-induced nuclear accumulation overrode insulin-induced nuclear exclusion of FoxO1a. Despite a slightly enhanced FoxO DNA binding activity in DEM-exposed cells, expression of FoxO-regulated genes (glucose 6-phosphatase, selenoprotein P) was downregulated, indicating that nuclear accumulation does not necessarily coincide with enhanced transcription factor activity. To test for an effect of DEM on organismal stress resistance, we exposed C. elegans roundworms to the thiol depletor. Survival in the presence of the redox cycler paraquat was significantly increased following exposure to DEM, implying that DEM pre-exposure induced cellular resistance against oxidative stress. Furthermore, in DEM-exposed C. elegans populations expressing a GFP-tagged version of the C. elegans FoxO ortholog, DAF-16, numbers of worms with predominantly nuclear DAF-16 increased - in line with the findings from HepG2 cells. In keeping with the known function of DAF-16 in stress resistance, C. elegans life span was elevated upon exposure to DEM in a concentration-dependent manner. A maximum extension of life span and deceleration of aging was achieved at 100 µ? of DEM. In summary, exposure to DEM caused a modulation of FoxO subcellular localization in both HepG2 cells and C. elegans roundworms, followed by a modulation of life span and stress resistance in C. elegans.