The implications of APOBEC3-mediated C-to-U RNA editing for human disease.

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans.

The Role of C-to-U RNA Editing in Human Biodiversity.

Intra-organism biodiversity is thought to arise from epigenetic modification of our constituent genes and post-translational modifications after mRNA is translated into proteins. We have found that post-transcriptional modification, also known as RNA editing, is also responsible for a significant amount of our biodiversity, substantively expanding this story. The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosines to uracils (C>U) in specific stem-loop structures.1,2 We used RNAsee (RNA site editing evaluation), a tool developed to predict the locations of APOBEC3A/G RNA editing sites, to determine whether known single nucleotide polymorphisms (SNPs) in DNA could be replicated in RNA via RNA editing. About 4.5% of non-synonymous SNPs which result in C>U changes in RNA, and about 5.4% of such SNPs labelled as pathogenic, were identified as probable sites for APOBEC3A/G editing. This suggests that the variant proteins created by these DNA mutations may also be created by transient RNA editing, with the potential to affect human health. Those SNPs identified as potential APOBEC3A/G-mediated RNA editing sites were disproportionately associated with cardiovascular diseases, digestive system diseases, and musculoskeletal diseases. Future work should focus on common sites of RNA editing, any variant proteins created by these RNA editing sites, and the effects of these variants on protein diversity and human health. Classically, our biodiversity is thought to come from our constitutive genetics, epigenetic phenomenon, transcriptional differences, and post-translational modification of proteins. Here, we have shown evidence that RNA editing, often stimulated by environmental factors, could account for a significant degree of the protein biodiversity leading to human disease. In an era where worries about our changing environment are ever increasing, from the warming of our climate to the emergence of new diseases to the infiltration of microplastics and pollutants into our bodies, understanding how environmentally sensitive mechanisms like RNA editing affect our own cells is essential.

Impact of Maternal Ketogenic Diet on NLRP3 Inflammasome Response in the Offspring Brain.

The effects of maternal diet on the neuroimmune responses of the offspring remain to be elucidated. We investigated the impact of maternal ketogenic diet (KD) on the NLRP3 inflammasome response in the offspring's brain. C57BL/6 female mice were randomly allocated into standard diet (SD) and ketogenic diet (KD) groups for 30 days. After mating, the presence of sperm in the vaginal smear was considered day 0 of pregnancy, and female mice continued their respective diets during pregnancy and the lactation period. Following birth, pups were further allocated into two groups and given either LPS or intraperitoneal saline on postnatal (PN) days 4, 5 and 6; they were sacrificed on PN11 or PN21. Neuronal densities were significantly lower globally in the KD group when compared to the SD group at PN11. Neuronal density in the prefrontal cortex (PFC) and dentate gyrus (DG) regions were also significantly lower in the KD group when compared to the SD group at PN21. Following administration of LPS, the decrease in the neuronal count was more prominent in the SD group when compared to the KD group in the PFC and DG regions at PN11 and PN21. NLRP3 and IL-1ß were higher in the KD group than in the SD group at PN21 in the PFC, CA1 and DG regions, and were significantly lower in the DG region of the KD group especially when compared to the SD group following LPS. Results of our study reveal that maternal KD negatively affects the offspring's brain in the mouse model. The effects of KD exhibited regional variations. On the other hand, in the presence of KD exposure, NLRP3 expression after LPS injection was lower in the DG and CA1 areas but not in the PFC when compared to SD group. Further experimental and clinical studies are warranted to elucidate the molecular mechanisms underlying the impact of antenatal KD exposure and regional discrepancies on the developing brain.

Unusual B-Lymphoid Blastic Crisis as Initial Presentation of Chronic Myeloid Leukemia Imposes Diagnostic Challenges.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder, characterized by reciprocal translocation t(9,22) (q34; q11), leading to increased myeloid proliferation. Most cases are diagnosed in the chronic phase (CP). However, a minority of cases can be present in the blastic phase (BP). In most patients with CML-BP, the blasts have a myeloid phenotype, however, in 20-30% of cases, the blasts have a lymphoid phenotype, mostly a B-cell phenotype. It is challenging to differentiate CML B-lymphoblastic phase (CML-BLP) from Ph + primary B-acute lymphoblastic leukemia (B-ALL) especially when the CML-BLP is the initial presentation of the disease, which is uncommon. We report here an unusual case of CML-BLP as an initial presentation of the disease without typical CML morphological findings. This case demonstrates diagnostic challenges and emphasizes the importance of an integrated approach using morphology, multiparametric flow cytometry, cytogenetic studies, and molecular studies to render an accurate diagnosis.

Succinate dehydrogenase inversely regulates red cell distribution width and healthy life span in chronically hypoxic mice.

Increased red cell distribution width (RDW), which measures erythrocyte mean corpuscular volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increased erythrocyte numbers, hemoglobin, and RDW, while reducing longevity in male mice. Compound heterozygous knockout (hKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc, and Sdhd reduced Sdh subunit protein levels, reduced RDW, and increased healthy life span compared with WT mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the SD of MCV showed the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites showed the largest increase in succinate with elevated succinate/fumarate and succinate/oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy life span in hypoxia and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.

Ofatumumab plus HyperCVAD/HD-MA induction leads to high rates of minimal residual disease negativity in patients with newly diagnosed mantle cell lymphoma: Results of a phase 2 study.

BACKGROUND: Ofatumumab is a humanized type 1 anti-CD20 monoclonal antibody. Preclinical studies show improved complement-mediated cytotoxicity (CMC) compared to rituximab in mantle cell lymphoma (MCL). This study evaluates the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL. METHODS: In this single-arm phase 2 study, 37 patients were treated with the combination of O-HyperCVAD for 4 or 6 cycles, followed by high dose chemotherapy and autologous stem cell transplant. Primary objectives were overall response rate (ORR) and complete response (CR) rate at the end of therapy. Secondary objectives included minimal residual disease (MRD) negativity, progression-free survival (PFS), and overall survival (OS). RESULTS: Median age was 60 years; ORR was 86% and 73% achieved a CR by modified Cheson criteria. The MRD negativity rate was 78% after 2 cycles of therapy, increasing to 96% at the end of induction; median PFS and OS were 45.5 months and 56 months, respectively. Achieving a post-induction CR by both imaging and flow cytometry was associated with improved PFS and OS. Early MRD negativity (post-2 cycles) was also associated with an improved PFS but not OS. There were 3 deaths while on therapy, and grades 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients. CONCLUSION: The addition of ofatumumab to HyperCVAD/HD-MA led to high rates of MRD negativity by flow cytometry in patients with newly diagnosed MCL. Achieving a CR post-induction by both imaging and flow cytometry is associated with improved overall survival.

RNA editing enzyme APOBEC3A promotes pro-inflammatory M1 macrophage polarization.

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1ß and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages.

Oxygen exposure in early life activates NLRP3 inflammasome in mouse brain.

Despite widely known detrimental effects on the developing brain, supplemental oxygen is still irreplaceable in the management of newborn infants with respiratory distress. Identifying downstream mechanisms underlying oxygen toxicity is a key step for development of new neuroprotective strategies. Main purpose of this study is to investigate whether NLRP3 inflammasome activation has a role in the pathogenesis of hyperoxia-induced preterm brain injury. C57BL6 pups were randomly divided into either a hyperoxia group (exposed to 90 % oxygen from birth until postnatal day 7) or control group (maintained in room air; 21 % O2). At postnatal day 7, all animals were sacrificed. Immunohistochemical examination revealed that hyperoxic exposure for seven days resulted in a global increase in NLRP3 and IL-1ß immunopositive cells in neonatal mouse brain (p ≤ 0.001). There was a significant rise in Caspase-1 positive cell count in prefrontal and parietal area in the hyperoxia group when compared with controls (p ≤ 0.001). Western blot analysis of brain tissues showed elevated NLRP3, IL-1ß and Caspase-1 protein levels in the hyperoxia group when compared with controls (p ≤ 0.001). To the best of our knowledge, this is the first study that investigates an association between hyperoxia and establishment of NLRP3 inflammasome in preterm brain.

Factors preventing materno-fetal transmission of SARS-CoV-2.

Although many pregnant women have been infected by coronavirus, the presence of intrauterine vertical transmission has not been conclusively reported yet. What prevents this highly contagious virus from reaching the fetus? Is it only the presence of a strong placental barrier, or is it the natural absence of the some receptor that the viruses use for transmission? We, therefore, need to comprehensively understand the mechanism of action of the mammalian epithelial barriers located in two different organs with functional similarity. The barriers selected as potential targets by SARS-CoV-2 are the alveolo-capillary barrier (ACB), and the syncytio-capillary barrier (SCB). Caveolae are omega-shaped structures located on the cell membrane. They consist of caveolin-1 protein (Cav-1) and are involved in the internalisation of some viruses. By activating leukocytes and nuclear factor-κB, Cav-1 initiates inflammatory reactions. The presence of more than one Cav-1 binding sites on coronavirus is an important finding supporting the possible relationship between SARS-CoV-2-mediated lung injury. While the ACB cells express Cav-1 there is no caveolin expression in syncytiotrophoblasts. In this short review, we will try to explain our hypothesis that the lack of caveolin expression in the SCB is one of the most important physiological mechanisms that prevents vertical transmission of SARS-CoV-2. Since the physiological Cav-1 deficiency appears to prevent acute cell damage treatment algorithms could potentially be developed to block this pathway in the non-pregnant population affected by SARS-CoV-2.

Combating sars-cov-2 through lipoxins, proteasome, caveolin and nuclear factor-κb pathways in non-pregnant and pregnant populations.

It can be misleading to think that the new severe acute respiratory syndrome coronavirus (SARS-CoV2) which has a very strong mutation and adaptation capabilities, uses only the angiotensin-converting enzyme II (ACE2) pathway to reach target cells. Despite all the precautions taken, the pandemic attack continues and the rapid increase in the number of deaths suggest that this virus has entered the cell through different pathways and caused damage through different mechanisms. The main reason why the ACE2 pathway comes to the fore in all scientific studies is that this receptor is located at the entry point of basic mechanisms that provide alveolo-capillary homeostasis. SARS-CoV-2 has to use nuclear factor-κB (NF-kB), caveloae, clathrin, lipoxin, serine protease and proteasome pathways in addition to ACE2 to enter the target cell and initiate damage. For this reason, while new drug development studies are continuing, in order to be beneficial to patients in their acute period, it is imperative that we are able to come up with drugs that activate or inhibit these pathways and are currently in clinical use. It is also critical that we adopt these new pathways to the treatment of pregnant women affected by SARS-CoV-2, based on the scientific data we use to treat the general population.

Is European Medicines Agency (EMA) sepsis criteria accurate for neonatal sepsis diagnosis or do we need new criteria?

BACKGROUND: Currently, there is a lack of clear definition for neonatal sepsis. The Pediatric Committee of the European Medicines Agency (EMA) developed consensus criteria to ensure a standardization for neonatal sepsis definition. However, there is no evidence supporting the accuracy of the EMA sepsis criteria in neonatal sepsis diagnosis. The main objective of this study was to evaluate the diagnostic accuracy of EMA sepsis criteria for proven neonatal sepsis. METHODS: A multicenter prospective cohort study was conducted from October 2015 to November 2018. Infants with a gestational age over 34th weeks, diagnosed with clinical sepsis and received antibiotics according to the EMA criteria or experienced neonatologists' opinion were included. Blood culture or multiplex real time-PCR or 16S-rRNA positive infants were accepted as "proven sepsis". The predictive performance of EMA criteria for proven sepsis was evaluated by sensitivity, specificity, accuracy, and area under the curve measures of receiver operator characteristic curves. Data-mining methods were used for further analysis. RESULTS: Among the 245 included infants, the EMA criteria were positive in 97 infants (39.6%), while proven sepsis was diagnosed in 113 infants (46.1%). The sensitivity, specificity, and accuracy of the EMA criteria for proven sepsis were 44.2% (95%CI: 34.9-53.9), 64.4% (95%CI: 55.6-72.5), 55.1% (95%CI: 46.6-59.4) respectively. None of the clinical and laboratory parameters had sufficient performance individually in terms of sensitivity, specificity and accuracy measures. The diagnostic performance was similar when different clinical findings were added to the EMA sepsis criteria or assessment of the score was interpreted in different ways. CONCLUSIONS: Results highlighted that clinician opinion and standard laboratory tests are limited in the neonatal sepsis diagnosis. The EMA criteria also did not efficiently meet the diagnostic accuracy measures for neonatal sepsis. A predictive sepsis definition and rapid bedside point-of care tests are urgently needed.

Mitochondrial hypoxic stress induces widespread RNA editing by APOBEC3G in natural killer cells.

BACKGROUND: Protein recoding by RNA editing is required for normal health and evolutionary adaptation. However, de novo induction of RNA editing in response to environmental factors is an uncommon phenomenon. While APOBEC3A edits many mRNAs in monocytes and macrophages in response to hypoxia and interferons, the physiological significance of such editing is unclear. RESULTS: Here, we show that the related cytidine deaminase, APOBEC3G, induces site-specific C-to-U RNA editing in natural killer cells, lymphoma cell lines, and, to a lesser extent, CD8-positive T cells upon cellular crowding and hypoxia. In contrast to expectations from its anti-HIV-1 function, the highest expression of APOBEC3G is shown to be in cytotoxic lymphocytes. RNA-seq analysis of natural killer cells subjected to cellular crowding and hypoxia reveals widespread C-to-U mRNA editing that is enriched for genes involved in mRNA translation and ribosome function. APOBEC3G promotes Warburg-like metabolic remodeling in HuT78 T cells under similar conditions. Hypoxia-induced RNA editing by APOBEC3G can be mimicked by the inhibition of mitochondrial respiration and occurs independently of HIF-1α. CONCLUSIONS: APOBEC3G is an endogenous RNA editing enzyme in primary natural killer cells and lymphoma cell lines. This RNA editing is induced by cellular crowding and mitochondrial respiratory inhibition to promote adaptation to hypoxic stress.

Comparison of INTERGROWTH-21 and Fenton growth standards to assess size at birth and extrauterine growth in very preterm infants.

AIM: This study aimed to compare the recently published prescriptive INTERGROWTH-21st standards with commonly used intrauterine based Fenton growth standards in terms of birth size classification and extrauterine growth restriction (EUGR) incidence in a sample of very preterm infants. METHODS: The anthropometric measures of preterm infants born before 32 weeks of gestation at the Dokuz Eylul University Hospital during the period from January 2012 to February 2016 were obtained at birth, at the 36th gestational weeks or at the time of discharge. Birth and growth data were presented as percentiles according to the two reference standards. RESULTS: A total of 248 infants with mean gestational age of 29.1 ± 2.1 weeks were included. The small for gestational age (SGA) rate was significantly higher (12 versus 15%, p = .004) and the EUGR rate was significantly lower (40.2 versus 31.5%, p < .001) with the INTERGROWTH-21st charts compared with the Fentons'. Twenty-four per cent of the infants who were accepted as SGA according to the INTERGROWTH-21st standards were appropriate for gestational age (AGA) according to the Fenton preterm growth charts. However, these newly identified SGA infants according to the Intergrowth-21st standards did not have increased risks of early morbidities. Furthermore, 77% of the cases who had EUGR due to the Fenton standards were categorized as EUGR when evaluated using the INTERGROWTH-21st standards. CONCLUSIONS: Results indicated that almost one out of every five cases assessed as EUGR according to Fenton standards was within the normal interval according to Intergrowth standards. On the contrary, one out of every four cases assessed as SGA according to the INTERGROWTH-21st standards was within the normal interval according to Fentons'. These differences observed with INTERGROWTH-21st standards may affect in-hospital and postdischarge nutrition plan of these vulnerable infants. However, new standards are needed to be evaluated against currently used ones before they are implemented and further studies should be conducted to evaluate the functional impact of these differences on long-term outcomes including neurologic and cardio-metabolic morbidities.

Stem-loop structure preference for site-specific RNA editing by APOBEC3A and APOBEC3G.

APOBEC3A and APOBEC3G cytidine deaminases inhibit viruses and endogenous retrotransposons. We recently demonstrated the novel cellular C-to-U RNA editing function of APOBEC3A and APOBEC3G. Both enzymes deaminate single-stranded DNAs at multiple TC or CC nucleotide sequences, but edit only a select set of RNAs, often at a single TC or CC nucleotide sequence. To examine the specific site preference for APOBEC3A and -3G-mediated RNA editing, we performed mutagenesis studies of the endogenous cellular RNA substrates of both proteins. We demonstrate that both enzymes prefer RNA substrates that have a predicted stem-loop with the reactive C at the 3'-end of the loop. The size of the loop, the nucleotides immediately 5' to the target cytosine and stability of the stem have a major impact on the level of RNA editing. Our findings show that both sequence and secondary structure are preferred for RNA editing by APOBEC3A and -3G, and suggest an explanation for substrate and site-specificity of RNA editing by APOBEC3A and -3G enzymes.

RNA Editing in Pathogenesis of Cancer.

Several adenosine or cytidine deaminase enzymes deaminate transcript sequences in a cell type or environment-dependent manner by a programmed process called RNA editing. RNA editing enzymes catalyze A>I or C>U transcript alterations and have the potential to change protein coding sequences. In this brief review, we highlight some recent work that shows aberrant patterns of RNA editing in cancer. Transcriptome sequencing studies reveal increased or decreased global RNA editing levels depending on the tumor type. Altered RNA editing in cancer cells may provide a selective advantage for tumor growth and resistance to apoptosis. RNA editing may promote cancer by dynamically recoding oncogenic genes, regulating oncogenic gene expression by noncoding RNA and miRNA editing, or by transcriptome scale changes in RNA editing levels that may affect innate immune signaling. Although RNA editing markedly increases complexity of the cancer cell transcriptomes, cancer-specific recoding RNA editing events have yet to be discovered. Epitranscriptomic changes by RNA editing in cancer represent a novel mechanism contributing to sequence diversity independently of DNA mutations. Therefore, RNA editing studies should complement genome sequence data to understand the full impact of nucleic acid sequence alterations in cancer. Cancer Res; 77(14); 3733-9. ©2017 AACR.

A prospective cohort study on the prediction of fetal distress and neonatal status with arterial and venous Doppler measurements in appropriately grown term fetuses.

PURPOSE: To assess the predictive power of the cerebro-placental ratio (CPR) and the venous-arterial index (VAI) for the development of intrapartum fetal distress (FD) and neonatal intensive care unit (NICU) admission. METHODS: Fetal umbilical artery, middle cerebral artery and umbilical vein Doppler measurements were obtained before the active phase of labor in 311 singleton pregnancies at ≥37 weeks. A continuous electronic fetal monitorization was applied, and an umbilical cord blood sample was obtained for each participant. FD and NICU admission were the primary outcomes. RESULTS: Labor was concluded as uncomplicated spontaneous vaginal delivery (SVD) in 261 (83.9%) cases. The 22 (7.1%) FD cases were subdivided into FD with NICU admission (n: 7; 2.3%) and without NICU admission (n: 15; 4.8%). Six out of 7 (85.8%) FD with NICU admission cases were from nulliparous pregnancies. The combinatory indices (VAI and CPR) reached the highest sensitivity (31.8%) and negative predictive value (94.7%). None of the fetuses, distressed or non-distressed, with CPR ≤ 10th percentile was born with a cord pH < 7.20. CONCLUSION: FD frequency was increased in fetuses with a low CPR or low VAI. However, the Doppler patterns were heterogeneous in both subgroups: FD with and without NICU admission. FD seems to be a common endpoint of different circulatory-metabolic disturbances. Parity affects the FD frequency in a manner related but not limited to fetal arterial and venous circulation. Low CPR could be a part of the adaptive mechanisms providing metabolic preparedness for hypoxic episodes.

Neurodevelopmental Outcome of Children with Congenital Hypothyroidism Diagnosed in a National Screening Program in Turkey.

OBJECTIVE: To study the factors affecting a neurodevelopmental status of children with congenital hypothyroidism, diagnosed on national screening program. METHODS: The study was performed in the Pediatric Endocrinology Department of Dr. Behcet Uz Children's Hospital between May 2012 and May 2013. Children with congenital hypothyroidism, aged between 24 and 36 months, diagnosed by national screening program were included in the study group. Healthy subjects at the same age group consisted of the control group. For the neurodevelopmental evaluation, Bayley Scale of Infant Development- II (BSID-II) was used. Factors possibly effective on neurodevelopment were evaluated. RESULTS: 42 patients and 40 healthy children (mean (SD) age, 29.4 (3.7) and 29.2 (3.5), respectively were included in the study. The mean MDI score [92.6 (7.07) vs 97.1 (9.69), P=0.14)] and the mean PDI score [97.8 (15.68) vs 99.1 (10.57), P=0.66)] in the study group and control group were not significantly different. Among the patient, 4.6% and 4.7% children were moderately retarded as per the MDI scores and PPI scores, respectively. The sex, socioeconomic status, birth weight, screening levels of TSH, severity of the congenital hypothyroidism, initiation time and the dosage of thyroid hormone replacement, length of the normalization period of TSH, and adherence to treatment were not found to affect the MDI and PDI scores of the patients. CONCLUSION: Some children with congenital hypothyrodism may have mild to moderate neurodevelopmental retardation, despite the early diagnosis and treatment, and thus need to be under regular follow-up for neurodevelopmental status.

Mitochondrial complex II regulates a distinct oxygen sensing mechanism in monocytes.

Mutations in mitochondrial complex II (succinate dehydrogenase; SDH) genes predispose to paraganglioma tumors that show constitutive activation of hypoxia responses. We recently showed that SDHB mRNAs in hypoxic monocytes gain a stop codon mutation by APOBEC3A-mediated C-to-U RNA editing. Here, we test the hypothesis that inhibition of complex II facilitates hypoxic gene expression in monocytes using an integrative experimental approach. By RNA sequencing, we show that specific inhibition of complex II by atpenin A5 in normoxic conditions mimics hypoxia and induces hypoxic transcripts as well as APOBEC3A-mediated RNA editing in human monocytes. Myxothiazol, a complex III inhibitor, has similar effects in normoxic monocytes. Atpenin A5 partially inhibits oxygen consumption, and neither hypoxia nor atpenin A5 in normoxia robustly stabilizes hypoxia-inducible factor (HIF)-1α in primary monocytes. Several earlier studies in transformed cell lines suggested that normoxic stabilization of HIF-1α explains the persistent expression of hypoxic genes upon complex II inactivation. On the contrary, we find that atpenin A5 antagonizes the stabilization of HIF-1α and reduces hypoxic gene expression in transformed cell lines. Accordingly, compound germline heterozygosity of mouse Sdhb/Sdhc/Sdhd null alleles blunts chronic hypoxia-induced increases in hemoglobin levels, an adaptive response mainly regulated by HIF-2α. In contrast, atpenin A5 or myxothiazol does not reduce hypoxia-induced gene expression or RNA editing in monocytes. These results reveal a novel role for mitochondrial respiratory inhibition in induction of the hypoxic transcriptome in monocytes and suggest that inhibition of complex II activates a distinct hypoxia signaling pathway in a cell-type specific manner.

Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes.

APOBEC3A cytidine deaminase induces site-specific C-to-U RNA editing of hundreds of genes in monocytes exposed to hypoxia and/or interferons and in pro-inflammatory macrophages. To examine the impact of APOBEC3A overexpression, we transiently expressed APOBEC3A in HEK293T cell line and performed RNA sequencing. APOBEC3A overexpression induces C-to-U editing at more than 4,200 sites in transcripts of 3,078 genes resulting in protein recoding of 1,110 genes. We validate recoding RNA editing of genes associated with breast cancer, hematologic neoplasms, amyotrophic lateral sclerosis, Alzheimer disease and primary pulmonary hypertension. These results highlight the fundamental impact of APOBEC3A overexpression on human transcriptome by widespread RNA editing.

The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing.