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RNA Biology Jan 2024In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the...
In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the APOBEC family of enzymes, mostly APOBEC1. RNA editing has a plethora of biological functions, and its deregulation has been associated with various human disorders. While the large-scale detection of A-to-I is quite straightforward using the Illumina RNAseq technology, the identification of C-to-U events is a non-trivial task. This difficulty arises from the rarity of such events in eukaryotic genomes and the challenge of distinguishing them from background noise. Direct RNA sequencing by Oxford Nanopore Technology (ONT) permits the direct detection of Us on sequenced RNA reads. Surprisingly, using ONT reads from wild-type (WT) and APOBEC1-knock-out (KO) murine cell lines as well as in vitro synthesized RNA without any modification, we identified a systematic error affecting the accuracy of the Cs call, thereby leading to incorrect identifications of C-to-U events. To overcome this issue in direct RNA reads, here we introduce a novel machine learning strategy based on the isolation Forest (iForest) algorithm in which C-to-U editing events are considered as sequencing anomalies. Using in vitro synthesized and human ONT reads, our model optimizes the signal-to-noise ratio improving the detection of C-to-U editing sites with high accuracy, over 90% in all samples tested. Our results suggest that iForest, known for its rapid implementation and minimal memory requirements, is a promising tool to denoise ONT reads and reliably identify RNA modifications.
Topics: Mice; Animals; Humans; RNA; RNA Editing; Base Sequence; APOBEC Deaminases; Mammals; Sequence Analysis, RNA
PubMed: 38090878
DOI: 10.1080/15476286.2023.2290843 -
European Journal of Clinical... Feb 2024Despite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein,...
PURPOSE
Despite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions.
METHODS
Sanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity.
RESULTS
The findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups.
CONCLUSIONS
These results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.
Topics: Humans; HIV-1; vif Gene Products, Human Immunodeficiency Virus; Cytidine Deaminase; HIV Infections; African People; APOBEC-3G Deaminase
PubMed: 38072879
DOI: 10.1007/s10096-023-04728-0 -
International Journal of Molecular... Nov 2023Identifying and understanding genetic factors that influence the propagation of the human respiratory syncytial virus (RSV) can lead to health benefits and possibly...
Identifying and understanding genetic factors that influence the propagation of the human respiratory syncytial virus (RSV) can lead to health benefits and possibly augment recent vaccine approaches. We previously identified a p53/immune axis in which the tumor suppressor p53 directly regulates the expression of immune system genes, including the seven members of the APOBEC3 family of DNA cytidine deaminases (A3), which are innate immune sentinels against viral infections. Here, we examined the potential p53 and A3 influence in RSV infection, as well as the overall p53-dependent cellular and p53/immune axis responses to infection. Using a paired p53 model system of p53+ and p53- human lung tumor cells, we found that RSV infection activates p53, leading to the altered p53-dependent expression of , , and along with p53 site-specific binding. Focusing on A3G because of its 10-fold-greater p53 responsiveness to RSV, the overexpression of can reduce RSV viral replication and syncytial formation. We also observed that RSV-infected cells undergo p53-dependent apoptosis. The study was expanded to globally address at the transcriptional level the p53/immune axis response to RSV. Nearly 100 genes can be directly targeted by the p53/immune axis during RSV infection based on our p53BAER analysis (Binding And Expression Resource). Overall, we identify A3G as a potential p53-responsive restriction factor in RSV infection. These findings have significant implications for RSV clinical and therapeutic studies and other p53-influenced viral infections, including using p53 adjuvants to boost the response of genes.
Topics: Humans; APOBEC-3G Deaminase; Cytidine Deaminase; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Tumor Suppressor Protein p53; Virus Replication
PubMed: 38069117
DOI: 10.3390/ijms242316793 -
BMC Pulmonary Medicine Dec 2023Small cell transformation was one mechanism by which EGFR-mutation NSCLC acquired resistance after tyrosine kinase inhibitors (TKIs) treatment. A few reports of small...
Small cell transformation was one mechanism by which EGFR-mutation NSCLC acquired resistance after tyrosine kinase inhibitors (TKIs) treatment. A few reports of small cell transformation occurred in other oncogene-driven lung cancers. We found the first case of transformation of a RET-rearranged lung adenocarcinoma to SCLC after selpercatinib, a novel highly selective RET TKIs. Whole-exome sequencing (WES) was used to explore alteration in gene expression in tumor tissue at initial diagnosis and after transformation into small cell carcinoma. We found that transformed into SCLC tumor tissue had inactivation of RB1 and TP53, with RET fusion was still present. In addition, the APOBEC family of cytidine deaminases appeared amplification. Although RET rearrangement still existed, using another RET TKIs was ineffective, and etoposide plus platinum might be an effective rescue treatment.
Topics: Humans; Lung Neoplasms; Exome Sequencing; ErbB Receptors; Protein Kinase Inhibitors; Small Cell Lung Carcinoma; Adenocarcinoma of Lung; Mutation; Proto-Oncogene Proteins c-ret
PubMed: 38057798
DOI: 10.1186/s12890-023-02799-5 -
Molecular Therapy. Nucleic Acids Dec 2023Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at... (Review)
Review
Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.
PubMed: 38028200
DOI: 10.1016/j.omtn.2023.102062 -
BioRxiv : the Preprint Server For... Nov 2023Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We...
Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We have shown that MMTV-encoded Rem protein inhibits proviral mutagenesis by the Apobec enzyme, activation-induced cytidine deaminase (AID) during viral replication in BALB/c mice. To further study the role of Rem , we have infected C57BL/6 (B6) mice with a superantigen-independent lymphomagenic strain of MMTV (TBLV-WT) or a mutant strain (TBLV-SD) that is defective in Rem and its cleavage product Rem-CT. Unlike MMTV, TBLV induced T-cell tumors in µMT mice, indicating that mature B cells, which express the highest AID levels, are not required for TBLV replication. Compared to BALB/c, B6 mice were more susceptible to TBLV infection and tumorigenesis. The lack of Rem expression accelerated B6 tumorigenesis at limiting doses compared to TBLV-WT in either wild-type B6 or AID-deficient mice. However, unlike proviruses from BALB/c mice, high-throughput sequencing indicated that proviral G-to-A or C-to-T changes did not significantly differ in the presence and absence of Rem expression. stimulation showed higher levels of mA3 relative to AID in B6 compared to BALB/c splenocytes, but effects of agonists differed in the two strains. RNA-Seq revealed increased transcripts related to growth factor and cytokine signaling in TBLV-SD-induced tumors relative to those from TBLV-WT, consistent with a third Rem function. Thus, Rem-mediated effects on tumorigenesis in B6 mice are independent of Apobec-mediated proviral hypermutation.
PubMed: 37961113
DOI: 10.1101/2023.11.02.565355 -
Science Advances Nov 2023Mutational signatures represent a genomic footprint of endogenous and exogenous mutational processes through tumor evolution. However, their functional impact on the...
Mutational signatures represent a genomic footprint of endogenous and exogenous mutational processes through tumor evolution. However, their functional impact on the proteome remains incompletely understood. We analyzed the protein-coding impact of single-base substitution (SBS) signatures in 12,341 cancer genomes from 18 cancer types. Stop-gain mutations (SGMs) (i.e., nonsense mutations) were strongly enriched in SBS signatures of tobacco smoking, APOBEC cytidine deaminases, and reactive oxygen species. These mutational processes alter specific trinucleotide contexts and thereby substitute serines and glutamic acids with stop codons. SGMs frequently affect cancer hallmark pathways and tumor suppressors such as , , and . Tobacco-driven SGMs in lung cancer correlate with smoking history and highlight a preventable determinant of these harmful mutations. APOBEC-driven SGMs are enriched in YTCA motifs and associate with expression. Our study exposes SGM expansion as a genetic mechanism by which endogenous and carcinogenic mutational processes directly contribute to protein loss of function, oncogenesis, and tumor heterogeneity.
Topics: Humans; Mutation; Neoplasms; Cytidine Deaminase; APOBEC Deaminases; Tobacco Smoking
PubMed: 37922356
DOI: 10.1126/sciadv.adh3083 -
Science (New York, N.Y.) Nov 2023Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa....
Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.
Topics: Animals; Humans; Africa, Central; Africa, Western; APOBEC Deaminases; Disease Outbreaks; Mpox (monkeypox); Monkeypox virus; Mutation; Phylogeny; Viral Zoonoses; RNA Editing
PubMed: 37917680
DOI: 10.1126/science.adg8116 -
Nature Communications Oct 2023Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper...
Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper replication, especially at head-on transcription-replication conflicts (TRCs). Here we show that TRCs trigger a DNA Damage Response (DDR) involving the chromatin network to prevent genome instability. Depletion of the key chromatin factors INO80, SMARCA5 and MTA2 results in TRCs, fork stalling and R-loop-mediated DNA damage which mostly accumulates at S/G2, while histone H3 Ser10 phosphorylation, a mark of chromatin compaction, is enriched at TRCs. Strikingly, TRC regions show increased mutagenesis in cancer cells with signatures of homologous recombination deficiency, transcription-coupled nucleotide excision repair (TC-NER) and of the AID/APOBEC cytidine deaminases, being predominant at head-on collisions. Thus, our results support that the chromatin network prevents R-loops and TRCs from genomic instability and mutagenic signatures frequently associated with cancer.
Topics: Humans; Chromatin; DNA Replication; Transcription, Genetic; Mutagenesis; DNA Damage; Genomic Instability; Neoplasms; Histone Deacetylases; Repressor Proteins
PubMed: 37898641
DOI: 10.1038/s41467-023-42653-0 -
BMC Bioinformatics Oct 2023Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites....
BACKGROUND
Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.
RESULTS
We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.
CONCLUSIONS
FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE .
Topics: RNA Editing; Workflow; RNA; RNA-Seq; Binding Sites; APOBEC-1 Deaminase
PubMed: 37784060
DOI: 10.1186/s12859-023-05452-4