RESUMO
The APOBEC/AID family is known for its mutator activity, and recent evidence also supports the potential impact of ADARs. Furthermore, the mutator impacts of APOBEC/ADAR mutations have not yet been investigated. Assessment of pancancer TCGA exomes identified enriched somatic variants among exomes with nonsynonymous APOBEC1, APOBEC3B, APOBEC3C, ADAR, and ADARB1 mutations, compared to exomes with synonymous ones. Principal component (PC) analysis reduced the number of potential players to eight in cancer exomes/genomes, and to five in cancer types. Multivariate regression analysis was used to assess the impact of the PCs on each COSMIC mutational signature among pancancer exomes/genomes and particular cancers, identifying several novel links, including SBS17b, SBS18, and ID7 mainly determined by APOBEC1 mRNA levels; SBS40, ID1, and ID2 by age; SBS3 and SBS16 by APOBEC3A/APOBEC3B mRNA levels; ID5 and DBS9 by DNA repair/replication (DRR) defects; and SBS7a-d, SBS38, ID4, ID8, ID13, and DBS1 by ultraviolet (UV) radiation/ADARB1 mRNA levels. APOBEC/ADAR mutations appeared to potentiate the impact of DRR defects on several mutational signatures, and some factors seemed to inversely affect certain signatures. These findings potentially implicate certain APOBEC/ADAR mutations/mRNA levels in distinct mutational signatures, particularly APOBEC1 mRNA levels in aging-related signatures and ADARB1 mRNA levels in UV radiation-related signatures.
Assuntos
Adenosina Desaminase , Envelhecimento , Mutação , RNA Mensageiro , Proteínas de Ligação a RNA , Raios Ultravioleta , Humanos , Raios Ultravioleta/efeitos adversos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Envelhecimento/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Neoplasias/genética , ExomaRESUMO
Cisplatin (CP) induces acute kidney injury (AKI) whereby proximal tubules undergo regulated necrosis. Repair is almost complete after a single dose. We now demonstrate a role for Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec-1) that is prominently expressed at the interface between acute and chronic kidney injury (CKD), in the recovery from AKI. Apobec-1 knockout (KO) mice exhibited greater mortality than in wild type (WT) and more severe AKI in both CP- and unilateral ischemia reperfusion (IR) with nephrectomy. Specifically, plasma creatinine (pCr) 2.6 ± 0.70 mg/dL for KO, n = 10 and 0.16 ± 0.02 for WT, n = 6, p < 0.0001 in CP model and 1.34 ± 0.22 mg/dL vs 0.75 ± 0.06, n = 5, p < 0.05 in IR model. The kidneys of Apobec-1 KO mice showed increased necrosis, increased expression of KIM-1, NGAL, RIPK1, ASCL4 and increased lipid accumulation compared to WT kidneys (p < 0.01). Neutrophils and activated T cells were both increased, while macrophages were reduced in kidneys of Apobec-1 KO animals. Overexpression of Apobec-1 in mouse proximal tubule cells protected against CP-induced cytotoxicity. These findings suggest that Apobec-1 mediates critical pro-survival responses to renal injury and increasing Apobec-1 expression could be an effective strategy to mitigate AKI.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Camundongos , Animais , Desaminase APOBEC-1/metabolismo , Cisplatino/efeitos adversos , Cisplatino/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Rim/metabolismo , Necrose/metabolismo , Camundongos Knockout , Traumatismo por Reperfusão/metabolismo , Camundongos Endogâmicos C57BLRESUMO
CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes. Here, we develop optimized C-to-T base editing systems for the generation of precise loss- or gain-of-function alleles in Drosophila and identify temperature as a crucial parameter for efficiency. We find that a variant of the widely used APOBEC1 deaminase has attenuated activity at 18° to 29°C and shows considerable dose-dependent toxicity. In contrast, the temperature-tolerant evoCDA1 domain mediates editing of typically more than 90% of alleles and is substantially better tolerated. Furthermore, formation of undesired mutations is exceptionally rare in Drosophila compared to other species. The predictable editing outcome, high efficiency, and product purity enables near homogeneous induction of STOP codons or alleles encoding protein variants in vivo. Last, we demonstrate how optimized expression enables conditional base editing in marked cell populations. This work substantially facilitates creation of precise alleles in Drosophila and provides key design parameters for developing efficient base editing systems in other ectothermic species.
Assuntos
Drosophila , Edição de Genes , Drosophila/genética , Edição de Genes/métodos , Animais , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Inativação GênicaRESUMO
Cytidine (C) to Uridine (U) RNA editing is a post-transcription modification that is involved in diverse biological processes. APOBEC1 (A1) catalyzes the conversion of C-to-U in RNA, which is important in regulating cholesterol metabolism through its editing activity on ApoB mRNA. However, A1 requires a cofactor to form an "editosome" for RNA editing activity. A1CF and RBM47, both RNA-binding proteins, have been identified as cofactors that pair with A1 to form editosomes and edit ApoB mRNA and other cellular RNAs. SYNCRIP is another RNA-binding protein that has been reported as a potential regulator of A1, although it is not directly involved in A1 RNA editing activity. Here, we describe the identification and characterization of a novel cofactor, RBM46 (RNA-Binding-Motif-protein-46), that can facilitate A1 to perform C-to-U editing on ApoB mRNA. Additionally, using the low-error circular RNA sequencing technique, we identified novel cellular RNA targets for the A1/RBM46 editosome. Our findings provide further insight into the complex regulatory network of RNA editing and the potential new function of A1 with its cofactors.
Assuntos
Desaminase APOBEC-1 , Edição de RNA , Proteínas de Ligação a RNA , Uridina , Humanos , Desaminase APOBEC-1/metabolismo , Desaminase APOBEC-1/genética , Apolipoproteínas B/metabolismo , Apolipoproteínas B/genética , Citidina/metabolismo , Citidina/genética , Células HEK293 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Uridina/metabolismo , Uridina/genéticaRESUMO
Although there have been some recent cell and animal experiments indicating that expression of the gene encoding apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) is closely related to cancer, it still lacks pan-cancer analysis. Here we analyzed the potential carcinogenic role of APOBEC3B in 33 tumors based on The Cancer Genome Atlas (TCGA). APOBEC3B was highly expressed in most tumors and weakly expressed in a few. Differences in expression level were significantly correlated with the pathological tumor stage and prognosis of affected patients. The high-frequency APOBEC3B changes were principally mutations and amplifications in some tumors, such as uterine corpus endometrial carcinomas or cutaneous melanomas. In testicular germ cell tumors and invasive breast carcinomas, APOBEC3B expression and CD8+ T lymphocyte counts were correlated. In other cancers, such as human papilloma virus (HPV)-related head and neck squamous cell carcinomas or esophageal adenocarcinomas, there was also cancer-associated fibroblast infiltration. The APOBEC3B enzyme acts in the mitochondrial respiratory electron transport chain and in oxidative phosphorylation. This first pan-cancer study provides a comprehensive understanding of the multiple roles of APOBEC3B in different tumor types.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Desaminase APOBEC-1/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Domínio Catalítico , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Neoplasias Esofágicas/genética , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
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.
Assuntos
Desaminase APOBEC-1/metabolismo , Neoplasias/patologia , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Vertebrados/crescimento & desenvolvimento , Desaminase APOBEC-1/genética , Animais , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) enzyme family in humans has 11 members with diverse functions in metabolism and immunity [...].
Assuntos
Desaminase APOBEC-1/genética , Vírus de DNA/imunologia , Imunidade Inata , Desaminase APOBEC-1/classificação , Desaminase APOBEC-1/metabolismo , Animais , Vírus de DNA/metabolismo , Humanos , Camundongos , Edição de RNARESUMO
The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.
Assuntos
Edição de Genes/métodos , Edição de RNA , Desaminase APOBEC-1/metabolismo , Adenosina Desaminase/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , HumanosRESUMO
Metastasis is inevitable in about 30% of patients with primary renal cell carcinoma after nephrectomy treatment. APOBEC1 complementation factor (A1CF), an RNA binding protein, participates in tumor progressions such as growth, apoptosis, differentiation, and invasion. Here, we explored biological functions of A1CF and provided a new insight into renal cell carcinoma metastasis. Wound healing assay was conducted to detect migration in A1CF overexpression and knockdown stable cell lines. Quantitative PCR and western blot assays were utilized to test transcriptional and translation levels of A1CF and SMAD3 in A1CF overexpression and knockdown renal carcinoma cells. Nuclear and cytoplasmic protein separation assays were conducted to evaluate the subcellular distribution of A1CF and SMAD3. Immunoprecipitation assay was conducted to detect the interaction between A1CF and SMAD3. Our study demonstrated A1CF overexpression facilitated cell migration in renal carcinoma cells. A1CF deficiency downregulated expression of SMAD3, Snail1, and N-cadherin. In addition, A1CF promoted nucleus translocation of SMAD3 and interacted with SMAD3. SMAD3 knockdown attenuated cell migration induced by A1CF overexpression. Our study suggested A1CF facilitated cell migration by promoting nucleus translocation of SMAD3 in renal cell carcinoma cells.
Assuntos
Desaminase APOBEC-1/metabolismo , Transporte Ativo do Núcleo Celular , Carcinoma de Células Renais/patologia , Movimento Celular , Neoplasias Renais/patologia , Proteína Smad3/metabolismo , Western Blotting , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunoprecipitação , Neoplasias Renais/metabolismo , Reação em Cadeia da PolimeraseRESUMO
As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses. IMPORTANCE Most of the emergences of new virus infections originate from the cross-species transmission of viruses. The fact that some virus infections are strictly specific for the host species indicates that certain "species barriers" in the hosts restrict cross-species jump of viruses, while viruses have evolutionary acquired their own "arms" to overcome/antagonize/neutralize these hurdles. Therefore, understanding of the molecular mechanism leading to successful cross-species viral transmission is crucial for considering the menus of the emergence of novel pathogenic viruses. In the field of retrovirology, APOBEC3-Vif interaction is a well-studied example of the battles between hosts and viruses. Here, we determined the sequences of 11 novel feline APOBEC3Z3 genes and demonstrated that all 18 different feline APOBEC3Z3 proteins tested exhibit anti-feline immunodeficiency virus (FIV) activity. Our comprehensive investigation focusing on the interplay between feline APOBEC3 and FIV Vif can be a clue to elucidate the scenarios of the cross-species transmissions of FIVs in felids.
Assuntos
Desaminase APOBEC-1/metabolismo , Produtos do Gene vif/metabolismo , Vírus da Imunodeficiência Felina/metabolismo , Infecções por Lentivirus/transmissão , Animais , Gatos , Linhagem Celular , Células HEK293 , Especificidade de Hospedeiro/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Infecções por Lentivirus/patologia , Panthera , Replicação Viral/fisiologiaRESUMO
APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity but rely on extraction of the RNA from tissues and cells. More recently, the need to visualize the RNA editing process directly in live cells have led to the development of fluorescence-based tools. These assays detect RNA editing through reporters whose editing causes a change in cellular localization or a change in fluorescent properties. Here we review the available assays to quantify RNA editing, and we present the protocol for cytofluorimetric analysis using a double-fluorescent reporter.
Assuntos
Desaminase APOBEC-1/genética , Biologia Computacional/métodos , Citidina/genética , Edição de RNA/genética , RNA Mensageiro/genética , Frações Subcelulares/metabolismo , Uridina/genética , Desaminase APOBEC-1/metabolismo , Citidina/química , Genes Reporter , Células HEK293 , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Mensageiro/metabolismo , Uridina/químicaRESUMO
Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.
Assuntos
Sistemas CRISPR-Cas/genética , Citosina/metabolismo , DNA Glicosilases , Edição de Genes/métodos , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Adenina/metabolismo , Animais , Pareamento de Bases/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Citidina Desaminase , Reparo do DNA/genética , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Escherichia coli/genética , Guanina/metabolismo , Ratos , Uracila-DNA GlicosidaseRESUMO
Many APOBEC cytidine deaminase members are known to induce 'off-target' cytidine deaminations in 5'TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.
Assuntos
Desaminase APOBEC-1/antagonistas & inibidores , DNA de Cadeia Simples/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Replicação A/metabolismo , Desaminase APOBEC-1/metabolismo , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Citidina/metabolismo , Dano ao DNA , Replicação do DNA , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/química , Desaminação , Difusão Facilitada , Histonas/análise , Humanos , Pulmão/citologia , Pulmão/embriologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/genéticaRESUMO
The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2nd-3rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/genética , Desaminase APOBEC-1/metabolismo , Citidina Desaminase/metabolismo , Análise Mutacional de DNA , Detecção Precoce de Câncer , Exoma , Genética , Centro Germinativo/patologia , Humanos , Modelos Lineares , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Proteínas/metabolismo , Edição de RNA , RNA Mensageiro , Análise de Célula ÚnicaRESUMO
Cytosine base editors (CBEs) enable efficient, programmable reversion of Tâ¢A to Câ¢G point mutations in the human genome. Recently, cytosine base editors with rAPOBEC1 were reported to induce unguided cytosine deamination in genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 with either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B [wt, R54Q], or PpAPOBEC1 [wt, H122A, R33A]) that display comparable DNA on-target editing frequencies, whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the transcriptome, and up to a 45-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Further, no enrichment of genome-wide Câ¢G to Tâ¢A edits are observed in mammalian cells following transfection of mRNA encoding five of these next-generation editors. Taken together, these next-generation CBEs represent a collection of base editing tools for applications in which minimized off-target and high on-target activity are required.
Assuntos
Citosina/metabolismo , DNA/genética , Edição de Genes , RNA/genética , Desaminase APOBEC-1/metabolismo , Citosina Desaminase/metabolismo , Replicação do DNA/genética , Desaminação , Genoma , Células HEK293 , Humanos , Mutagênese/genética , Transcrição Gênica , Transcriptoma/genéticaRESUMO
Hepatitis B virus (HBV) DNA is vulnerable to editing by human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. However, the distribution of APOBEC-induced mutations on HBV DNA is not well characterized. To this end, we obtained the HBV DNA sequence of HBV-infected individuals with and without hepatocellular carcinoma (HCC and non-HCC groups, respectively) from NCBI database and calculated the rapo values of APOBEC-induced TpCpWâTpKpW mutation prevalence in HBV DNA. The results showed that the APOBEC-induced mutations were mainly distributed in the minus strand of non-HCC-derived HBV DNA (rapo = 2.04), while the mutation on the plus-strand was weaker (rapo = 0.99). There were high APOBEC-induced mutation regions in the minus strand of HBV DNA 1 to 1000 nucleotides (nts) region and in the plus-strand of HBV DNA 1000 to 1500 nts region; the mutations in the 1 to 1000 nts region were mainly TpCpWâTpTpW mutation types (total T/G: 111/18) and a number of these were missense mutations (missense/synonymous: 35/94 in P gene, 17/15 in S gene, and 5/10 in X gene). The difference between minus to plus-strand rapo of HCC-derived HBV DNA (1.96) was greater than that of the non-HCC group (1.05). The minus-strand rapo of HCC-derived HBV DNA regions 1000 to1500nts and 1500 to 2000 nts (rapo = 4.2 and 4.2) was also higher than that of the same regions of non-HCC-derived HBV DNA (rapo = 1.2 and 1.1). Finally, the ratio of minus to plus-strand rapo was used to distinguish HCC-derived HBV DNA from non-HCC-derived HBV DNA. This study unraveled the distribution characteristics of APOBEC-induced mutations on double strands of HBV DNA from HCC and non-HCC samples. Our findings would help understand the mechanism of APOBECs on HBV DNA and may provide important insights for the screening of HCC.
Assuntos
Desaminase APOBEC-1/metabolismo , DNA Viral/genética , Genótipo , Vírus da Hepatite B/genética , Mutação , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/classificação , Humanos , Neoplasias Hepáticas/virologia , Análise de Sequência de DNARESUMO
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.
Assuntos
Desaminase APOBEC-1/metabolismo , Cromossomos de Mamíferos/genética , Mutação , Desaminase APOBEC-1/química , Desaminase APOBEC-1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Ativação Enzimática , Expressão Gênica , Camundongos , Filogenia , Edição de RNA , Especificidade por SubstratoRESUMO
Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.
Assuntos
Desaminase APOBEC-1/fisiologia , DNA Mitocondrial/química , Drosophila/genética , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Animais , Drosophila/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Modelos Genéticos , Mutação , Organismos Geneticamente ModificadosRESUMO
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.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Edição de RNA , Especificidade por Substrato/genética , Transcriptoma/genética , Desaminase APOBEC-1/química , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Animais , Sequência de Bases , Citosina/metabolismo , Desaminação , Células HEK293 , Células Hep G2 , Humanos , Mutação , RNA/química , RNA/metabolismo , RatosRESUMO
RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.