APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

A Long-Running Arms Race between APOBEC1 Genes and Retroviruses in Tetrapods.

Activation-induced cytidine deaminase/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) proteins are cytosine deaminases implicated in diverse biological functions. APOBEC1 (A1) proteins have long been thought to regulate lipid metabolism, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. Endogenous retroviruses (ERVs) document past retroviral infections and are ubiquitous within the vertebrate genomes. Here, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in ERVs, and interrogate the evolutionary arms race between A1 genes and ERVs across vertebrate species. We find that A1 genes are widely present in tetrapods, recurrently amplified and lost in certain lineages, suggesting that A1 genes might have originated during the early evolution of tetrapods. A1-mediated mutation footprints can be detected in ERVs across tetrapods. Moreover, A1 genes appear to have experienced episodic positive selection in many tetrapod lineages. Taken together, we propose that a long-running arms race between A1 genes and retroviruses might have persisted throughout the evolutionary course of tetrapods. IMPORTANCE APOBEC3 (A3) genes have been thought to function in defense against retroviruses, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. In this study, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in endogenous retroviruses (ERVs), and explore the evolutionary arms race between A1 genes and ERVs across vertebrate species. We found A1 proteins originated during the early evolution of tetrapods, and detected the footprints of A1-induced hypermutations in retroviral fossils. A1 genes appear to have experienced pervasive positive selection in tetrapods. Our study indicates a long-running arms race between A1 genes and retroviruses taking place throughout the evolutionary course of tetrapods.

APOBEC-1 Complementation Factor: From RNA Binding to Cancer.

BACKGROUND: APOBEC-1 complementation factor (A1CF) and Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1 (APOBEC-1) constitute the minimal proteins necessary for the editing of apolipoprotein B (apoB) mRNA in vitro. Unlike APOBEC-1 and apoB mRNA, the ubiquitous expression of A1CF in human tissues suggests its unique biological significance, with various factors such as protein kinase C, thyroid hormones, and insulin regulating the activity and expression of A1CF. Nevertheless, few studies have provided an overview of this topic. OBJECTIVE: We conducted a literature review to describe the molecular mechanisms of A1CF and its relevance to human diseases. METHOD: In the PubMed database, we used the keywords 'A1CF' and 'APOBEC-1 complementation factor' to collect peer-reviewed articles published in English from 2000 to 2023. Two authors independently reviewed the articles and reached the consensus. RESULT: After reviewing 127 articles, a total of 61 articles that met the inclusion criteria were included in the present review. Studies revealed that A1CF is involved in epigenetic regulation of reproductive cells affecting embryonic development, and that it is closely associated with the occurrence of gout due to its editing properties on apoB. A1CF can also affect the process of epithelial-mesenchymal transition in renal tubular epithelial cells and promote liver regeneration by controlling the stability of IL-6 mRNA, but no influence on cardiac function was found. Furthermore, increasing evidence suggests that A1CF may promote the occurrence and development of breast cancer, lung cancer, renal cell carcinoma, hepatocellular carcinoma, endometrial cancer, and glioma. CONCLUSION: This review clarifies the association between A1CF and other complementary factors and their impact on the development of human diseases, aiming to provide guidance for further research on A1CF, which can help treat human diseases and promote health.

Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Integrated genomic and molecular characterization of cervical cancer.

Cervical cancer remains one of the leading causes of cancer-related deaths worldwide. Here we report the extensive molecular characterization of 228 primary cervical cancers, one of the largest comprehensive genomic studies of cervical cancer to date. We observed notable APOBEC mutagenesis patterns and identified SHKBP1, ERBB3, CASP8, HLA-A and TGFBR2 as novel significantly mutated genes in cervical cancer. We also discovered amplifications in immune targets CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2), and the BCAR4 long non-coding RNA, which has been associated with response to lapatinib. Integration of human papilloma virus (HPV) was observed in all HPV18-related samples and 76% of HPV16-related samples, and was associated with structural aberrations and increased target-gene expression. We identified a unique set of endometrial-like cervical cancers, comprised predominantly of HPV-negative tumours with relatively high frequencies of KRAS, ARID1A and PTEN mutations. Integrative clustering of 178 samples identified keratin-low squamous, keratin-high squamous and adenocarcinoma-rich subgroups. These molecular analyses reveal new potential therapeutic targets for cervical cancers.

DART-seq: an antibody-free method for global m6A detection.

N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.

Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice.

Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N4 GATT and N4 CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.

Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver.

Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf-/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf-/- mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (ARLKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (ARIKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in ARIKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

RNA binding motif 47 (RBM47): emerging roles in vertebrate development, RNA editing and cancer.

RNA-binding proteins (RBPs) are critical players in the post-transcriptional regulation of gene expression and are associated with each event in RNA metabolism. The term 'RNA-binding motif' (RBM) is assigned to novel RBPs with one or more RNA recognition motif (RRM) domains that are mainly involved in the nuclear processing of RNAs. RBM47 is a novel RBP conserved in vertebrates with three RRM domains whose contributions to various aspects of cellular functions are as yet emerging. Loss of RBM47 function affects head morphogenesis in zebrafish embryos and leads to perinatal lethality in mouse embryos, thereby assigning it to be an essential gene in early development of vertebrates. Its function as an essential cofactor for APOBEC1 in C to U RNA editing of several targets through substitution for A1CF in the A1CF-APOBEC1 editosome, established a new paradigm in the field. Recent advances in the understanding of its involvement in cancer progression assigned RBM47 to be a tumor suppressor that acts by inhibiting EMT and Wnt/[Formula: see text]-catenin signaling through post-transcriptional regulation. RBM47 is also required to maintain immune homeostasis, which adds another facet to its regulatory role in cellular functions. Here, we review the emerging roles of RBM47 in various biological contexts and discuss the current gaps in our knowledge alongside future perspectives for the field.

Epac1 (Exchange Protein Directly Activated by cAMP 1) Upregulates LOX-1 (Oxidized Low-Density Lipoprotein Receptor 1) to Promote Foam Cell Formation and Atherosclerosis Development.

OBJECTIVE: The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model (LTe) was generated by crossing Epac1-/- mice with atherosclerosis-prone LDb mice lacking both Ldlr and Apobec1. Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining in LTe animals compared with LDb littermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis. CONCLUSIONS: Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells.

Loss of APOBEC1 RNA-editing function in microglia exacerbates age-related CNS pathophysiology.

Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.

RNA Binding Proteins as Drivers and Therapeutic Target Candidates in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinomas (PDAC) belong to the most frequent and most deadly malignancies in the western world. Mutations in KRAS and TP53 along with some other frequent polymorphisms occur almost universally and are likely to be responsible for tumor initiation. However, these mutations cannot explain the heterogeneity in therapeutic responses observed in PDAC patients, which limits efficiency of current therapeutic strategies. Instead, recent classifications of PDAC tumor samples are based on transcriptomics data and thus include information about epigenetic, transcriptomic, and post-transcriptomic deregulations. RNA binding proteins (RBPs) are important post-transcriptional regulators involved in every aspect of the RNA life cycle and thus considerably influence the transcriptome. In this study, we systematically investigated deregulated expression, prognostic value, and essentiality reported for RBPs in PDAC or PDAC cancer models using publicly available data. We identified 44 RBPs with suggested oncogenic potential. These include various proteins, e.g., IGF2 mRNA binding proteins (IGF2BPs), with reported tumor-promoting roles. We further characterized these RBPs and found common patterns regarding their expression, interaction, and regulation by microRNAs. These analyses suggest four prime candidate oncogenic RBPs with partially validated target potential: APOBEC1, IGF2BP1 and 3, and OASL.

Mouse APOBEC1 cytidine deaminase can induce somatic mutations in chromosomal DNA.

BACKGROUND: APOBEC1 (A1) enzymes are cytidine deaminases involved in RNA editing. In addition to this activity, a few A1 enzymes have been shown to be active on single stranded DNA. As two human ssDNA cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B) and related enzymes across the spectrum of placental mammals have been shown to introduce somatic mutations into nuclear DNA of cancer genomes, we explored the mutagenic threat of A1 cytidine deaminases to chromosomal DNA. RESULTS: Molecular cloning and expression of various A1 enzymes reveal that the cow, pig, dog, rabbit and mouse A1 have an intracellular ssDNA substrate specificity. However, among all the enzymes studied, mouse A1 appears to be singular, being able to introduce somatic mutations into nuclear DNA with a clear 5'TpC editing context, and to deaminate 5-methylcytidine substituted DNA which are characteristic features of the cancer related mammalian A3A and A3B enzymes. However, mouse A1 activity fails to elicit formation of double stranded DNA breaks, suggesting that mouse A1 possess an attenuated nuclear DNA mutator phenotype reminiscent of human A3B. CONCLUSIONS: At an experimental level mouse APOBEC1 is remarkable among 12 mammalian A1 enzymes in that it represents a source of somatic mutations in mouse genome, potentially fueling oncogenesis. While the order Rodentia is bereft of A3A and A3B like enzymes it seems that APOBEC1 may well substitute for it, albeit remaining much less active. This modifies the paradigm that APOBEC3 and AID enzymes are the sole endogenous mutator enzymes giving rise to off-target editing of mammalian genomes.

The Scavenger Receptor CD68 Regulates Platelet Mediated Oxidized Low-Density Lipoprotein (oxLDL) Deposition in Atherosclerotic Vessels at an Early Stage of Atherosclerosis in LDLR-/-/ApoBec-/- Mice.

BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.

APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.

Within-Host Variations of Human Papillomavirus Reveal APOBEC Signature Mutagenesis in the Viral Genome.

Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied by the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here, we explored within-host genetic diversity of HPV by performing deep-sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52, and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) and were deep sequenced. After constructing a reference viral genome sequence for each specimen, nucleotide positions showing changes with >0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with various numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the trinucleotide context encompassing substituted bases revealed that TpCpN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions.IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep-sequencing analyses, we show for the first time a comprehensive snapshot of the within-host genetic diversity of high-risk HPVs during cervical carcinogenesis. Quasispecies harboring minor nucleotide variations in viral whole-genome sequences were extensively observed across different grades of CIN and cervical cancer. Among the within-host variations, C-to-T transitions, a characteristic change mediated by cellular APOBEC cytosine deaminases, were predominantly detected throughout the whole viral genome, most strikingly in low-grade CIN lesions. The results strongly suggest that within-host variations of the HPV genome are primarily generated through the interaction with host cell DNA-editing enzymes and that such within-host variability is an evolutionary source of the genetic diversity of HPVs.

CRISPR-Cas9D10A nickase-assisted base editing in the solvent producer Clostridium beijerinckii.

Clostridium beijerinckii is a potentially important industrial microorganism as it can synthesize valuable chemicals and fuels from various carbon sources. The establishment of convenient to use, effective gene tools with which the organism can be rapidly modified is essential if its full potential is to be realized. Here, we developed a genomic editing tool (pCBEclos) for use in C. beijerinckii based on the fusion of cytidine deaminase (Apobec1), Cas9 D10A nickase and uracil DNA glycosylase inhibitor (UGI). Apobec1 and UGI are guided to the target site where they introduce specific base-pair substitutions through the conversion of C·G to T·A. By appropriate choice of target sequence, these nucleotide changes are capable of creating missense mutation or null mutations in a gene. Through optimization of pCBEclos, the system derived, pCBEclos-opt, has been used to rapidly generate four different mutants in C. beijerinckii, in pyrE, xylR, spo0A, and araR. The efficiency of the system was such that they could sometimes be directly obtained following transformation, otherwise only requiring one single restreaking step. Whilst CRISPR-Cas9 nickase systems, such as pNICKclos2.0, have previously been reported in C. beijerinckii, pCBEclos-opt does not rely on homologous recombination, a process that is intrinsically inefficient in clostridia such as C. beijerinckii. As a consequence, bulky editing templates do not need to be included in the knockout plasmids. This both reduces plasmid size and makes their construction simpler, for example, whereas the assembly of pNICKclos2.0 requires six primers for the assembly of a typical knockout plasmid, pCBEclos-opt requires just two primers. The pCBEclos-opt plasmid established here represents a powerful new tool for genome editing in C. beijerinckii, which should be readily applicable to other clostridial species.