Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m5C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulates the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.

Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N6-methyladenosine (m6A), 5-methylcytosine (m5C), and 2'O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.IMPORTANCE The data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C, and Nm, suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication in cis, and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in trans Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression.

The mRNA modification N6-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor. Furthermore, the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines, providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line, overexpression of METTL3 and METTL14, or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably, these treatments had little effect on the immortalized cells. Together, these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally, we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3, METTL14 or ALKBH5 expression, suggesting a novel pathway for regulation of m6A levels under stress.

Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Accumulating data support a role for bioactive lipids as mediators of lipotixicity in cardiomyocytes. One class of these, the ceramides, constitutes a family of molecules that differ in structure and are synthesized by distinct enzymes, ceramide synthase (CerS)1-CerS6. Data support that specific ceramides and the enzymes that catalyze their formation play distinct roles in cell function. In a mouse model of diabetic cardiomyopathy, sphingolipid profiling revealed increases in not only the CerS5-derived ceramides but also in very long chain (VLC) ceramides derived from CerS2. Overexpression of CerS2 elevated VLC ceramides caused insulin resistance, oxidative stress, mitochondrial dysfunction, and mitophagy. Palmitate induced CerS2 and oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. Neither overexpression nor knockdown of CerS5 had any function in these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function. This concept was also supported by the observation that synthetic mitochondria-targeted ceramides led to mitophagy in a manner proportional to N-acyl chain length. Finally, blocking mitophagy exacerbated cell death. Taken together, our results support a model by which CerS2 and VLC ceramides have a distinct role in lipotoxicity, leading to mitochondrial damage, which results in subsequent adaptive mitophagy. Our data reveal a novel lipotoxic pathway through CerS2.-Law, B. A., Liao, X., Moore, K. S., Southard, A., Roddy, P., Ji, R., Szulc, Z., Bielawska, A., Schulze, P. C., Cowart, L. A. Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs.

Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m6A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m6A. Our results show hypoxia increases m6A content of poly(A)+ messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m6A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m6A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m6A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m6A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m6A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress.

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes.

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-ß inhibition.

BACKGROUND: Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as alcoholic cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM, and prior studies by this laboratory have associated this occurrence with elevated transforming growth factor-beta (TGF-ß) and activated fibroblasts (myofibroblasts). To date, there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. METHODS: Primary rat cardiac fibroblasts were cultured in the presence of ethanol (EtOH) and assayed for fibroblast activation by collagen gel contraction, alpha-smooth muscle actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I and III, and TGF-ß expression. The TGF-ß receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-ßII receptor (RbII) were used to assess the role of TGF-ß in the response of cardiac fibroblasts to EtOH. RESULTS: Treatment for cardiac fibroblasts with EtOH at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-ß. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-ß by SB 431542 and RbII attenuated the EtOH-induced fibroblast activation. CONCLUSIONS: EtOH treatment directly promotes cardiac fibroblast activation by stimulating TGF-ß release from fibroblasts. Inhibiting the action of TGF-ß decreases the fibrogenic effect induced by EtOH treatment. The results of this study support TGF-ß to be an important component in cardiac fibrosis induced by exposure to EtOH.

Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts.

AIMS: Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM. MAIN METHODS: Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling. KEY FINDINGS: Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype. SIGNIFICANCE: Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.

Alterations in cardiac structure and function in a murine model of chronic alcohol consumption.

Male, wild-type, FVB strain mice were fed a nutritionally complete liquid diet supplemented with 4% ethanol v/v over a time course of 1, 2, 4, 8, 12, and 14 weeks. Controls were offered an isocaloric liquid equivalent and pair fed with their ethanol counterparts. Changes in cardiac physiology were assessed at respective time points via echocardiography. Additionally, the use of histological techniques, mRNA analysis, apoptosis determination, and immunohistochemistry were employed to determine the functional and structural changes on the heart. Echocardiograph analysis revealed a compensatory phase that occurred early in the time course (1-8 weeks) and decompensation reverting toward heart failure at weeks 12 and 14. Throughout the study, an increase in cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis, TGF-ß, and the presence of α-SMA-positive cells were determined. A compensatory period in mice treated with ethanol occurred early followed by a transition to a dilated phenotype over time. A number of factors may be involved in this process including the activation of myofibroblasts and their fibrotic activities that is correlated with the presence of transforming growth factor beta.

Substance P induces adverse myocardial remodelling via a mechanism involving cardiac mast cells.

AIMS: Substance P and neurokinin A (NKA) are sensory nerve neuropeptides encoded by the TAC1 gene. Substance P is a mast cell secretagogue and mast cells are known to play a role in adverse myocardial remodelling. Therefore, we wondered whether substance P and/or NKA modulates myocardial remodelling via a mast cell-mediated mechanism. METHODS AND RESULTS: Volume overload was induced by aortocaval fistula in TAC1(-/-) mice and their respective wild types. Left ventricular internal diameter of wild-type (WT) fistulas increased by 31.9%; this was prevented in TAC1(-/-) mice (4.2%). Matrix metalloproteinase (MMP) activity was significantly increased in WT fistula mice and was prevented in TAC1(-/-) mice. Myocardial collagen volume fraction was decreased in WT fistula mice; this collagen degradation was not observed in the TAC1(-/-) group. There were no significant differences between any groups in tumour necrosis factor (TNF)-α or cell death. Cardiac mast cells were isolated from rat hearts and stimulated with substance P or NKA. We found that these cells degranulated only to substance P, via the neurokinin-1 receptor. To determine the effect of substance P on mast cells in vivo, volume overload was created in Sprague-Dawley rats treated with the NK-1 receptor antagonist L732138 (5 mg/kg/day) for a period of 3 days. L732138 prevented: (i) increases in cardiac mast cell density; (ii) increased myocardial TNF-α; and (iii) collagen degradation. CONCLUSIONS: Our studies suggest that substance P may be important in mediating adverse myocardial remodelling secondary to volume overload by activating cardiac mast cells, leading to increased TNF-α and MMP activation with subsequent degradation of the extracellular matrix.

A putative azoreductase gene is involved in the Shewanella oneidensis response to heavy metal stress.

The Shewanella oneidensis MR-1 gene SO3585, which is annotated as a putative flavin mononucleotide-dependent azoreductase, shares 28% sequence identity with Bacillus subtilis azoreductase and Pseudomonas putida ChrR, a soluble flavoprotein exhibiting chromate reductase activity. Reverse transcription polymerase chain reaction demonstrated that the SO3585 gene is co-transcribed with two downstream open reading frames: SO3586 (a glyoxalase family protein) and SO3587 (a predicted membrane-associated hypothetical protein). The transcriptional start site of the so3585 transcript was localized using 5' rapid amplification of complementary DNA ends analysis. To investigate the cellular function of SO3585, an in-frame deletion of the so3585 locus was generated in MR-1, and the phenotype of the resulting mutant was characterized. The so3585 deletion mutant was comparable to the parental strain in its ability to decolorize two sulfonated azo dyes (Orange II, Direct Blue 15) under aerobic conditions. By contrast, growth of the so3585 deletion mutant was sensitive to different exogenous transition heavy metals [Cr(VI), Cd(II), Cu(II), and Zn(II)], while the most severe growth deficiencies were observed in the presence of Cd(II) and Cu(II). In addition, the rate of extracellular chromate disappearance by the deletion strain was initially impaired, although both the so3585 mutant and MR-1 wild type reduced Cr(VI) within the same time period.