Assuntos
Adenosina Desaminase , Agamaglobulinemia , Terapia de Reposição de Enzimas , Humanos , Terapia de Reposição de Enzimas/métodos , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Agamaglobulinemia/tratamento farmacológico , Agamaglobulinemia/terapia , Agamaglobulinemia/genética , Resultado do Tratamento , Masculino , Feminino , Mutação/genética , Imunodeficiência Combinada SeveraRESUMO
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
Assuntos
Adenosina , Citosina , Edição de Genes , Distrofia Muscular de Duchenne , Citosina/metabolismo , Animais , Edição de Genes/métodos , Adenosina/metabolismo , Camundongos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Engenharia de Proteínas , Distrofina/genética , Distrofina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Modelos Animais de Doenças , Éxons/genética , Células HEK293 , Sistemas CRISPR-Cas , Proteínas de Escherichia coliRESUMO
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/metabolismoRESUMO
Expansion of CAG repeats in certain genes is a known cause of several neurodegenerative diseases, but exact mechanism behind this is not yet fully understood. It is believed that the double-stranded RNA regions formed by CAG repeats could be harmful to the cell. This study aimed to test the hypothesis that these RNA regions might potentially interfere with ADAR RNA editing enzymes, leading to the reduced A-to-I editing of RNA and activation of the interferon response. We studied induced pluripotent stem cells (iPSCs) derived from the patients with Huntington's disease or ataxia type 17, as well as midbrain organoids developed from these cells. A targeted panel for next-generation sequencing was used to assess editing in the specific RNA regions. Differentiation of iPSCs into brain organoids led to increase in the ADAR2 gene expression and decrease in the expression of protein inhibitors of RNA editing. As a result, there was increase in the editing of specific ADAR2 substrates, which allowed identification of differential substrates of ADAR isoforms. However, comparison of the pathology and control groups did not show differences in the editing levels among the iPSCs. Additionally, brain organoids with 42-46 CAG repeats did not exhibit global changes. On the other hand, brain organoids with the highest number of CAG repeats in the huntingtin gene (76) showed significant decrease in the level of RNA editing of specific transcripts, potentially involving ADAR1. Notably, editing of the long non-coding RNA PWAR5 was nearly absent in this sample. It could be stated in conclusion that in most cultures with repeat expansion, the hypothesized effect on RNA editing was not confirmed.
Assuntos
Adenosina Desaminase , Encéfalo , Diferenciação Celular , Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Organoides , Edição de RNA , Proteínas de Ligação a RNA , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Humanos , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Encéfalo/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Expansão das Repetições de TrinucleotídeosRESUMO
Background: COVID-19 vaccines are crucial for reducing the threat and burden of the pandemic on global public health, yet the epigenetic, especially RNA editing in response to the vaccines remains unelucidated. Results: Our current study performed an epitranscriptomic analysis of RNA-Seq data of 260 blood samples from 102 healthy and SARS-CoV-2 naïve individuals receiving different doses of the COVID-19 vaccine and revealed dynamic, transcriptome-wide adenosine to inosine (A-to-I) RNA editing changes in response to COVID-19 vaccines (RNA editing in response to COVID-19 vaccines). 5592 differential RNA editing (DRE) sites in 1820 genes were identified, with most of them showing up-regulated RNA editing and correlated with increased expression of edited genes. These deferentially edited genes were primarily involved in immune- and virus-related gene functions and pathways. Differential ADAR expression probably contributed to RNA editing in response to COVID-19 vaccines. One of the most significant DRE in RNA editing in response to COVID-19 vaccines was in apolipoprotein L6 (APOL6) 3' UTR, which positively correlated with its up-regulated expression. In addition, recoded key antiviral and immune-related proteins such as IFI30 and GBP1 recoded by missense editing was observed as an essential component of RNA editing in response to COVID-19 vaccines. Furthermore, both RNA editing in response to COVID-19 vaccines and its functions dynamically depended on the number of vaccine doses. Conclusion: Our results thus underscored the potential impact of blood RNA editing in response to COVID-19 vaccines on the host's molecular immune system.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Epigênese Genética , Edição de RNA , SARS-CoV-2 , Humanos , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Adenosina/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Transcriptoma , Adenosina Desaminase/genética , Masculino , Adulto , Inosina , FemininoRESUMO
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/imunologiaRESUMO
Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.
Assuntos
Adenosina Desaminase , RNA de Cadeia Dupla , Proteínas de Ligação a RNA , eIF-2 Quinase , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , eIF-2 Quinase/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Humanos , Animais , Camundongos , Ligação Proteica , Ativação Enzimática , Células A549 , Domínios ProteicosRESUMO
Adenosine deaminase 2 (ADA2) deficiency is an autosomal recessively inherited autoinflammatory disorder caused by loss-of-function mutations in the ADA2 gene. Although the pathogenesis involves the triggering of a proinflammatory cascade due to increased production of inflammatory cytokines such as tumor necrosis factor (TNF)-α and dysregulation of neutrophil extracellular trap formation resulting from an excess accumulation of extracellular adenosine, the pathogenetic mechanism still needs further clarification due to the broad clinical spectrum. In addition to the initially described vasculitis-related symptoms, hematological, immunological, and autoinflammatory symptoms are now well recognized. The diagnosis is made by demonstration of pathogenic variants of ADA2 with biallelic loss of function and identification of low plasma ADA2 catalytic activity. Currently, TNF-α inhibitors are the treatment of choice for controlling vasculitis manifestations and preventing strokes. However, in patients presenting with severe hematologic findings, TNF-α inhibitors are not the treatment of choice and hematopoietic stem cell transplantation has been shown to be successful in selected cases. Recombinant ADA2 protein and gene therapy are promising treatment modalities for the future. In conclusion, ADA2 deficiency has a broad phenotype and should be considered in the differential diagnosis of different clinical situations. In this review, we summarize the disease manifestations of ADA2 deficiency and available treatment options.
Assuntos
Adenosina Desaminase , Humanos , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/terapia , Agamaglobulinemia/genética , Transplante de Células-Tronco Hematopoéticas , Fenótipo , Terapia Genética/métodos , Gerenciamento Clínico , Fator de Necrose Tumoral alfa , Mutação , Imunodeficiência Combinada Severa , Doenças Hereditárias AutoinflamatóriasRESUMO
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/genéticaRESUMO
AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.
Assuntos
Adenosina Desaminase , Edição de RNA , Proteínas de Ligação a RNA , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Humanos , Uridina/metabolismo , Uridina/química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA/química , RNA/metabolismo , Citidina/química , Citidina/metabolismo , Modelos Moleculares , Células HEK293RESUMO
RNA base editing relies on the introduction of adenosine-to-inosine changes into target RNAs in a highly programmable manner in order to repair disease-causing mutations. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory phosphorylation and acetylation sites. We demonstrate the feasibility on more than 70 sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. For the JAK/STAT pathway, we demonstrate both, negative and positive regulation. To achieve high editing efficiency over a broad codon scope, we applied an improved version of the SNAP-ADAR tool. The transient nature of RNA base editing enables the comparably fast (hours to days), dose-dependent (thus partial) and reversible manipulation of regulatory sites, which is a key advantage over DNA (base) editing approaches. In summary, PTM interference might become a valuable field of application of RNA base editing.
Assuntos
Processamento de Proteína Pós-Traducional , Edição de RNA , Humanos , Fosforilação , Células HEK293 , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , RNA/metabolismo , RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , AcetilaçãoRESUMO
Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience.
Assuntos
Adenosina Desaminase , Homeostase , Músculo Liso Vascular , Miócitos de Músculo Liso , Animais , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Aorta/metabolismo , Aorta/patologia , Apoptose/genética , Fibrilina-1/genética , Fibrilina-1/metabolismo , Elastina/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
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 InsetoRESUMO
Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.
Assuntos
Adenosina Desaminase , Proteínas de Ligação a RNA , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proliferação de Células/genética , Mitose/genética , Animais , Replicação do DNA/genética , Tetraploidia , Genoma Humano , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Camundongos , Edição de RNA , Linhagem Celular TumoralRESUMO
Post-replicative DNA N6-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.
Assuntos
Adenina , Adenosina Desaminase , Lentivirus , Animais , Camundongos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenina/metabolismo , Adenina/análogos & derivados , Lentivirus/genética , Metilação de DNA/genética , DNA/genética , DNA/metabolismo , HumanosRESUMO
Patients with amyotrophic lateral sclerosis (ALS) do not develop oculomotor disturbances and vesicorectal dysfunction until end-stage disease owing to the survival of certain motor neurons (MNs), including oculomotor neurons and MNs within Onuf's nucleus. In sporadic ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated editing of GluA2 mRNA at the Q/R site is compromised in lower MNs. We previously developed genetically modified mice with a conditional knockout of ADAR2 in cholinergic neurons (ADAR2flox/flox/VAChT-Cre, Fast; AR2). These mice displayed slow and progressive lower motor neuron death with TAR DNA-binding protein 43 (TDP-43) pathology, attributable to insufficient editing at the GluA2 Q/R site due to ADAR2 deficiency. MN death was more common in fast-fatigable MNs owing to differential vulnerability under conditions of ADAR2 deficiency. Although facial and hypoglossal nerves were impaired in AR2 mice, cell death did not occur within the oculomotor nerve nucleus, as observed in patients with sporadic ALS. Since the basis for avoiding cystorectal damage in ALS is unknown, we compared the features of Onuf's nucleus MNs in 12-month-old AR2 mice with those in age-matched wild-type mice. Although the number of MNs was not significantly lower in AR2 mice, the neurons exhibited a shrunken morphology and TDP-43 pathology. Onuf's nucleus MNs could survive in an ADAR2-deficient state and mainly included fast fatigue-resistant (FR) and slow (S) MNs. In summary, FR and S MNs show increased resilience to ADAR2 deficiency, potentially participating in an important neuronal death avoidance mechanism in ALS.
Assuntos
Adenosina Desaminase , Esclerose Lateral Amiotrófica , Camundongos Knockout , Neurônios Motores , Proteínas de Ligação a RNA , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Adenosina Desaminase/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Neurônios Motores/patologia , Neurônios Motores/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Camundongos TransgênicosRESUMO
The F11 receptor (F11R) gene encoding junctional adhesion molecule A has been associated with gastric cancer (GC) and colorectal cancer (CRC), in which its role and regulation remain to be further elucidated. Recently F11R was also identified as a potential target of adenosine-to-inosine (A-to-I) mediated by the adenosine deaminases acting on RNA (ADARs). Herein, using RNA-Seq and experimental validation, our current study revealed an F11R RNA trinucleotide over-edited by ADAR, with its regulation of gene expression and clinical significance in four GC and three CRC cohorts. Our results found an over-edited AAA trinucleotide in an AluSg located in the F11R 3'-untranslated region (3'-UTR), which showed editing levels correlated with elevated ADAR expression across all GC and CRC cohorts in our study. Overexpression and knockdown of ADAR in GC and CRC cells, followed by RNA-Seq and Sanger sequencing, confirmed the ADAR-mediated F11R 3'-UTR trinucleotide editing, which potentially disrupted an RBM45 binding site identified by crosslinking immunoprecipitation sequencing (CLIP-seq) and regulated F11R expression in luciferase reporter assays. Moreover, the F11R trinucleotide editing showed promising predictive performance for diagnosing GC and CRC across GC and CRC cohorts. Our findings thus highlight both the potential biological and clinical significance of an ADAR-edited F11R trinucleotide in GC and CRC, providing new insights into its application as a novel diagnostic biomarker for both cancers.
Assuntos
Adenosina Desaminase , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Edição de RNA , Proteínas de Ligação a RNA , Neoplasias Gástricas , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Estudos de Coortes , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Masculino , FemininoRESUMO
PURPOSE OF REVIEW: In this review, an update is provided on the current knowledge and pending questions about human adenosine deaminase type 2 deficiency. Patients have vasculitis, immunodeficiency and some have bone marrow failure. Although the condition was described ten years ago, the pathophysiology is incompletely understood RECENT FINDINGS: Endothelial instability due to increased proinflammatory macrophage development is key to the pathophysiology. However, the physiological role of ADA2 is a topic of debate as it is hypothesized that ADA2 fulfils an intracellular role. Increasing our knowledge is urgently needed to design better treatments for the bone marrow failure. Indeed, TNFi treatment has been successful in treating DADA2, except for the bone marrow failure. Major advances have been made in our understanding of DADA2. More research is needed into the physiological role of ADA2.
Assuntos
Adenosina Desaminase , Peptídeos e Proteínas de Sinalização Intercelular , Humanos , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/terapia , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/terapia , Doenças Hereditárias AutoinflamatóriasRESUMO
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/metabolismoRESUMO
Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.