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1.
Cancer Med ; 13(17): e70143, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39267574

RESUMO

BACKGROUND: This study aimed to evaluate whether inosine enhances the efficacy of immune-checkpoint inhibitors in human malignant solid tumors. METHODS: This single-center, prospective, randomized, open-label study was conducted, from January 2021 to December 2022, in Beijing Friendship Hospital, Capital Medical University, and participants were randomly assigned (1:1) to either the inosine (trial) or non-inosine (control) group that received inosine (dosage: 0.2 g, three times/day) + PD-1/PD-L1 inhibitor or only PD-1/PD-L1 inhibitor ± targeted ± chemotherapy, respectively. Efficacy was assessed every 6 weeks (i.e., after every two-three treatment cycles). The primary endpoint was the objective response rate (ORR); the secondary endpoints were disease control rate, overall survival (OS), and progression-free survival (PFS). The trial was registered at ClinicalTrials.gov (NCT05809336). RESULTS: Among the 172 participants with advanced malignant solid tumors, 86 each were assigned to the inosine and non-inosine groups, wherein the median PFS (95% CI) was 7.00 (5.31-8.69) and 4.40 (3.10-5.70) months, respectively (hazard ratio [HR] 0.63; 95% CI 0.44-0.90, p = 0.011), and the ORR was 26.7% and 15.1%, respectively (p = 0.061). In the inosine and non-inosine groups, the median OS was not reached and was 29.67 (95% CI 17.40-41.94) months, respectively (HR 1.05 [95% CI 0.59-1.84], p = 0.874). Compared with the non-inosine group, the median PFS and ORR of the inosine group were significantly prolonged and improved in the multiple exploratory subgroup analyses. The safety analysis showed that Grades 3 and 4 adverse reactions occurred in 25 (29%) and 31 (36%) patients in the inosine and non-inosine groups, respectively, and tended to decrease in the inosine group compared with the non-inosine group. CONCLUSION: Inosine had a tendency to enhance the efficacy of immune-checkpoint inhibitors and reduced immunotherapy-related adverse reactions.


Assuntos
Inibidores de Checkpoint Imunológico , Inosina , Neoplasias , Humanos , Inosina/uso terapêutico , Masculino , Feminino , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/efeitos adversos , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Neoplasias/imunologia , Idoso , Estudos Prospectivos , Adulto , Intervalo Livre de Progressão , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Sinergismo Farmacológico
2.
Sci Adv ; 10(36): eadi9101, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39231215

RESUMO

A-to-I RNA editing is a cellular mechanism that generates transcriptomic and proteomic diversity, which is essential for neuronal and immune functions. It involves the conversion of specific adenosines in RNA molecules to inosines, which are recognized as guanosines by cellular machinery. Despite the vast number of editing sites observed across the animal kingdom, pinpointing critical sites and understanding their in vivo functions remains challenging. Here, we study the function of an evolutionary conserved editing site in Drosophila, located in glutamate-gated chloride channel (GluClα). Our findings reveal that flies lacking editing at this site exhibit reduced olfactory responses to odors and impaired pheromone-dependent social interactions. Moreover, we demonstrate that editing of this site is crucial for the proper processing of olfactory information in projection neurons. Our results highlight the value of using evolutionary conservation as a criterion for identifying editing events with potential functional significance and paves the way for elucidating the intricate link between RNA modification, neuronal physiology, and behavior.


Assuntos
Canais de Cloreto , Edição de RNA , Animais , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olfato/fisiologia , Olfato/genética , Comportamento Animal , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Inosina/metabolismo , Inosina/genética , Odorantes , Adenosina/metabolismo , Drosophila/genética
3.
RNA Biol ; 21(1): 29-45, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39256954

RESUMO

Adar-mediated adenosine-to-inosine (A-to-I) mRNA editing is a conserved mechanism that exerts diverse regulatory functions during the development, evolution, and adaptation of metazoans. The accurate detection of RNA editing sites helps us understand their biological significance. In this work, with an improved genome assembly of honeybee (Apis mellifera), we used a new orthology-based methodology to complement the traditional pipeline of (de novo) RNA editing detection. Compared to the outcome of traditional pipeline, we retrieved many novel editing sites in CDS that are deeply conserved between honeybee and other distantly related insects. The newly retrieved sites were missed by the traditional de novo identification due to the stringent criteria for controlling false-positive rate. Caste-specific editing sites are identified, including an Ile>Met auto-recoding site in Adar. This recoding was even conserved between honeybee and bumblebee, suggesting its putative regulatory role in shaping the phenotypic plasticity of eusocial Hymenoptera. In summary, we proposed a complementary approach to the traditional pipeline and retrieved several previously unnoticed CDS editing sites. From both technical and biological aspects, our works facilitate future researches on finding the functional editing sites and advance our understanding on the connection between RNA editing and the great phenotypic diversity of organisms.


Assuntos
Adenosina , Evolução Molecular , Inosina , Edição de RNA , Animais , Inosina/genética , Inosina/metabolismo , Abelhas/genética , Adenosina/metabolismo , Adenosina/genética , Sequência Conservada , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo
4.
Genes (Basel) ; 15(8)2024 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-39202357

RESUMO

Inosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the "bioinformatics for inosine" subject and we discuss future research avenues in the field.


Assuntos
Adenosina Desaminase , Biologia Computacional , Inosina , Edição de RNA , Inosina/metabolismo , Inosina/genética , Biologia Computacional/métodos , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Humanos , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
5.
BMC Genomics ; 25(1): 803, 2024 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-39187830

RESUMO

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is a co-/post-transcriptional modification introducing A-to-G variations in RNAs. There is extensive discussion on whether the flexibility of RNA editing exerts a proteomic diversification role, or it just acts like hardwired mutations to correct the genomic allele. Eusocial insects evolved the ability to generate phenotypically differentiated individuals with the same genome, indicating the involvement of epigenetic/transcriptomic regulation. METHODS: We obtained the genomes of 104 Hymenoptera insects and the transcriptomes of representative species. Comparative genomic analysis was performed to parse the evolutionary trajectory of a regulatory Ile > Met auto-recoding site in Adar gene. RESULTS: At genome level, the pre-editing Ile codon is conserved across a node containing all eusocial hymenopterans. At RNA level, the editing events are confirmed in representative species and shows considerable condition-specificity. Compared to random expectation, the editable Ile codon avoids genomic substitutions to Met or to uneditable Ile codons, but does not avoid mutations to other unrelated amino acids. CONCLUSIONS: The flexibility of Adar auto-recoding site in Hymenoptera is selectively maintained, supporting the flexible RNA editing hypothesis. We proposed a new angle to view the adaptation of RNA editing, providing another layer to explain the great phenotypical plasticity of eusocial insects.


Assuntos
Adenosina Desaminase , Adenosina , Evolução Molecular , Inosina , Edição de RNA , Animais , Inosina/metabolismo , Inosina/genética , Adenosina/metabolismo , Adenosina/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Filogenia , Insetos/genética , Himenópteros/genética , Transcriptoma , Genoma de Inseto
6.
PLoS One ; 19(8): e0307450, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39178184

RESUMO

Adenosine to inosine (A-to-I) RNA editing by ADAR1 has been implicated in maintaining self-tolerance, preventing autoimmunity, and mediating antiviral immunity. Foreign viral double-stranded RNA triggers rapid interferon response and activates ADAR1 in the host immune system. Emerging data points to a role of ADAR1 A-to-I editing in the inflammatory response associated with severe COVID-19 disease. We identify A-to-I editing events within human whole transcriptome data from SARS-CoV-2 infected individuals, non-infected individuals, and individuals with other viral illnesses from nasopharyngeal swabs. High levels of RNA editing in host cells are associated with low SARS-CoV-2 viral load (p = 9.27 E-06), suggesting an inhibitory effect of ADAR1 on viral infection. Additionally, we find differentially expressed genes associated with RNA-modifications and interferon response. Single cell RNA-sequencing analysis of SARS-CoV-2 infected nasopharyngeal swabs reveals that cytotoxic CD8 T cells upregulate ADAR1 in COVID-19 positive samples (p = 0.0269). We further reveal ADAR1 expression increases with CD4 and CD8 T cell activation, and knockdown of ADAR1 leads to apoptosis and aberrant IL-2 secretion. Together, our data suggests A-to-I RNA editing is required to maintain healthy homeostasis of activated T cells to combat SARS-CoV-2 infection.


Assuntos
Adenosina Desaminase , COVID-19 , Homeostase , Edição de RNA , Proteínas de Ligação a RNA , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/virologia , COVID-19/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Viral , Inosina/metabolismo , Adenosina/metabolismo , Ativação Linfocitária/imunologia
7.
Cell Biol Toxicol ; 40(1): 57, 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39060787

RESUMO

It is well established that sevoflurane exposure leads to widespread neuronal cell death in the developing brain. Adenosine deaminase acting on RNA-1 (ADAR1) dependent adenosine-to-inosine (A-to-I) RNA editing is dynamically regulated throughout brain development. The current investigation is designed to interrogate the contributed role of ADAR1 in developmental sevoflurane neurotoxicity. Herein, we provide evidence to show that developmental sevoflurane priming triggers neuronal pyroptosis, apoptosis and necroptosis (PANoptosis), and elicits the release of inflammatory factors including IL-1ß, IL-18, TNF-α and IFN-γ. Additionally, ADAR1-P150, but not ADAR1-P110, depresses cellular PANoptosis and inflammatory response by competing with Z-DNA/RNA binding protein 1 (ZBP1) for binding to Z-RNA in the presence of sevoflurane. Further investigation demonstrates that ADAR1-dependent A-to-I RNA editing mitigates developmental sevoflurane-induced neuronal PANoptosis. To restore RNA editing, we utilize adeno-associated virus (AAV) to deliver engineered circular ADAR-recruiting guide RNAs (cadRNAs) into cells, which is capable of recruiting endogenous adenosine deaminases to promote cellular A-to-I RNA editing. As anticipated, AAV-cadRNAs diminishes sevoflurane-induced cellular Z-RNA production and PANoptosis, which could be abolished by ADAR1-P150 shRNA transfection. Moreover, AAV-cadRNAs delivery ameliorates developmental sevoflurane-induced spatial and emotional cognitive deficits without influence on locomotor activity. Taken together, these results illustrate that ADAR1-P150 exhibits a prominent role in preventing ZBP1-dependent PANoptosis through A-to-I RNA editing in developmental sevoflurane neurotoxicity. Application of engineered cadRNAs to rectify the compromised ADAR1-dependent A-to-I RNA editing provides an inspiring direction for possible clinical preventions and therapeutics.


Assuntos
Adenosina Desaminase , Adenosina , Edição de RNA , Proteínas de Ligação a RNA , Sevoflurano , Animais , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Apoptose/efeitos dos fármacos , Inosina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Piroptose/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
8.
J Mol Evol ; 92(4): 488-504, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39012510

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing recodes the genetic information. Apart from diversifying the proteome, another tempting advantage of RNA recoding is to correct deleterious DNA mutation and restore ancestral allele. Solid evidences for beneficial restorative editing are very rare in animals. By searching for "convergent recoding" under a phylogenetic context, we proposed this term for judging the potential restorative functions of particular editing site. For the well-known mammalian Gln>Arg (Q>R) recoding site, its ancestral state in vertebrate genomes was the pre-editing Gln, and all 470 available mammalian genomes strictly avoid other three equivalent ways to achieve Arg in protein. The absence of convergent recoding from His>Arg, or synonymous mutations on Gln codons, could be attributed to the strong maintenance on editing motif and structure, but the absence of direct A-to-G mutation is extremely unexpected. With similar ideas, we found cases of convergent recoding in Drosophila genus, reducing the possibility of their restorative function. In summary, we defined an interesting scenario of convergent recoding, the occurrence of which could be used as preliminary judgements for whether a recoding site has a sole restorative role. Our work provides novel insights to the natural selection and evolution of RNA editing.


Assuntos
Adenosina , Códon , Evolução Molecular , Inosina , Filogenia , Edição de RNA , Edição de RNA/genética , Animais , Inosina/genética , Adenosina/genética , Adenosina/metabolismo , Códon/genética , Seleção Genética , Humanos , Drosophila/genética
9.
Genes (Basel) ; 15(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-39062643

RESUMO

Cancer is a complex and heterogeneous disease, in which a number of genetic and epigenetic changes occur in tumor onset and progression. Recent studies indicate that changes at the RNA level are also involved in tumorigenesis, such as adenosine-to-inosine (A-to-I) RNA editing. Here, we systematically investigate transcriptome-wide A-to-I editing events in a large number of samples from Non-Hodgkin lymphomas (NHLs). Using a computational pipeline that determines significant differences in editing level between NHL and normal samples at known A-to-I editing sites, we identify a number of differentially edited editing sites between NHL subtypes and normal samples. Most of the differentially edited sites are located in non-coding regions, and many such sites show a strong correlation between gene expression level and editing efficiency, indicating that RNA editing might have direct consequences for the cancer cell's aberrant gene regulation status in these cases. Moreover, we establish a strong link between RNA editing and NHL by demonstrating that NHL and normal samples and even NHL subtypes can be distinguished based on genome-wide RNA editing profiles alone. Our study establishes a strong link between RNA editing, cancer and aberrant gene regulation in NHL.


Assuntos
Linfoma não Hodgkin , Edição de RNA , Linfoma não Hodgkin/genética , Adenosina/genética , Inosina/genética , Análise de Sequência de RNA , Expressão Gênica , Perfilação da Expressão Gênica , Família Multigênica
10.
Bioorg Med Chem ; 110: 117837, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39013280

RESUMO

RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I34) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I34 between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.


Assuntos
Adenosina , Inosina , Edição de RNA , Inosina/metabolismo , Inosina/química , Adenosina/metabolismo , Adenosina/química , Adenosina/análise , Animais , Camundongos , Humanos , Endonucleases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
11.
Genome Biol ; 25(1): 173, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956576

RESUMO

BACKGROUND: RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.


Assuntos
Haplótipos , Humanos , Linhagem Celular Tumoral , Polimorfismo de Nucleotídeo Único , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Pulmonares/genética , Splicing de RNA , Inosina/metabolismo , Inosina/genética , Análise de Sequência de RNA , Adenocarcinoma de Pulmão/genética , Edição de RNA , Software
12.
Int J Mol Sci ; 25(13)2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39000531

RESUMO

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.


Assuntos
Adenosina Desaminase , Neoplasias da Mama , Edição de RNA , Proteínas de Ligação a RNA , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Feminino , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Inosina/metabolismo , Inosina/genética , Animais , Guanosina/metabolismo , Dano ao DNA
13.
Sci Rep ; 14(1): 14973, 2024 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-38951658

RESUMO

Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.


Assuntos
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidade por Substrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química
14.
J Clin Invest ; 134(17)2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-38954486

RESUMO

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and adenosine deaminase isoform switching. We found that A-to-I editing of antizyme inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I-edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.


Assuntos
Adenosina , Inosina , Edição de RNA , Animais , Camundongos , Inosina/metabolismo , Inosina/genética , Adenosina/metabolismo , Adenosina/genética , Humanos , Rim/metabolismo , Rim/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Endotoxemia/metabolismo , Endotoxemia/genética , Endotoxemia/patologia , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Masculino
15.
Genes (Basel) ; 15(7)2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-39062677

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.


Assuntos
Adenosina Desaminase , Edição de RNA , Proteínas de Ligação a RNA , Schizosaccharomyces , Schizosaccharomyces/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Edição de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Humanos , Especificidade por Substrato , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Adenosina/metabolismo , Adenosina/genética , Inosina/genética , Inosina/metabolismo
16.
Fly (Austin) ; 18(1): 2367359, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38889318

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed 'conserved editing with non-conserved recoding' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.


Assuntos
Adenosina , Drosophila , Inosina , Edição de RNA , Animais , Inosina/metabolismo , Inosina/genética , Drosophila/genética , Drosophila/metabolismo , Adenosina/metabolismo , Adenosina/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo
17.
PLoS Pathog ; 20(6): e1012238, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38843141

RESUMO

Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.


Assuntos
Adenosina Desaminase , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Adenosina/metabolismo , Adenosina/genética , Inosina/metabolismo , Inosina/genética , Fusarium/genética , Neurospora crassa/genética
18.
Viruses ; 16(6)2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38932237

RESUMO

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.


Assuntos
Vírus Chikungunya , Genoma Viral , Processamento Pós-Transcricional do RNA , RNA Viral , Transcriptoma , Vírus Chikungunya/genética , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Febre de Chikungunya/virologia , Inosina/metabolismo , Inosina/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Adenosina/metabolismo , Adenosina Desaminase
19.
Nucleic Acids Res ; 52(12): 6733-6747, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38828787

RESUMO

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.


Assuntos
Adenosina Desaminase , Edição de RNA , Ribose , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/química , Ribose/química , Ribose/metabolismo , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Metilação , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/química , Nucleosídeos/química , Nucleosídeos/metabolismo , RNA/metabolismo , RNA/química , Inosina/metabolismo , Inosina/química
20.
Virus Res ; 346: 199413, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38848818

RESUMO

The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.


Assuntos
Adenosina Desaminase , Fibroblastos , Inosina , Edição de RNA , Transcriptoma , Animais , Camundongos , Fibroblastos/virologia , Fibroblastos/metabolismo , Inosina/metabolismo , Inosina/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenosina/metabolismo , Adenosina/genética , Infecções por Reoviridae/virologia , Infecções por Reoviridae/genética , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reoviridae/genética , Reoviridae/fisiologia
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