Site-Directed RNA Editing in Vivo Can Be Triggered by the Light-Driven Assembly of an Artificial Riboprotein

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

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Wilms' Tumor Suppressor GeneWT1

Journal of the American Society of Nephrology ◽

10.1681/asn.v11suppl_2s106 ◽

2000 ◽

Vol 11 (suppl 2) ◽

pp. S106-S115 ◽

Cited By ~ 3

Author(s):

CHRISTIAN MROWKA ◽

ANDREAS SCHEDL

Keyword(s):

Tumor Suppressor ◽

Rna Editing ◽

Kidney Development ◽

Wilms Tumor ◽

Suppressor Gene ◽

Structural Features ◽

Genomic Locus ◽

Developmental Abnormalities ◽

Complex Process

Abstract.Normal development of the kidney is a highly complex process that requires precise orchestration of proliferation, differentiation, and apoptosis. In the past few years, a number of genes that regulate these processes, and hence play pivotal roles in kidney development, have been identified. The Wilms' tumor suppressor geneWT1has been shown to be one of these essential regulators of kidney development, and mutations in this gene result in the formation of tumors and developmental abnormalities such as the Denys-Drash and Frasier syndromes. A fascinating aspect of theWT1gene is the multitude of isoforms produced from its genomic locus. In this review, our current understanding of the structural features ofWT1, how they modulate the transcriptional and post-transcriptional activities of the protein, and how mutations affecting individual isoforms can lead to diseased kidneys is summarized. In addition, results from transgenic experiments, which have yielded important findings regarding the function of WT1in vivo, are discussed. Finally, data on the unusual feature of RNA editing ofWT1transcripts are presented, and the relevance of RNA editing for the normal functioning of the WT1 protein in the kidney is discussed.

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CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

Nature Biotechnology ◽

10.1038/s41587-021-01105-0 ◽

2022 ◽

Author(s):

Philipp Reautschnig ◽

Nicolai Wahn ◽

Jacqueline Wettengel ◽

Annika E. Schulz ◽

Ngadhnjim Latifi ◽

...

Keyword(s):

Rna Editing ◽

Guide Rnas

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In vivo dissection of cis-acting determinants for plastid RNA editing.

The EMBO Journal ◽

10.1002/j.1460-2075.1996.tb00885.x ◽

1996 ◽

Vol 15 (18) ◽

pp. 5052-5059 ◽

Cited By ~ 93

Author(s):

R. Bock ◽

M. Hermann ◽

H. Kössel

Keyword(s):

Rna Editing ◽

Cis Acting

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SUMO-1 Modification Alters ADAR1 Editing Activity

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0536 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5115-5126 ◽

Cited By ~ 75

Author(s):

Joana M.P. Desterro ◽

Liam P. Keegan ◽

Ellis Jaffray ◽

Ron T. Hay ◽

Mary A. O'Connell ◽

...

Keyword(s):

Rna Editing ◽

Nucleolar Localization ◽

Dense Fibrillar Component ◽

Wild Type ◽

Fibrillar Center ◽

Editing Activity ◽

Fibrillar Component ◽

Editing Enzyme

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

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MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

10.1101/829606 ◽

2019 ◽

Cited By ~ 5

Author(s):

Jeetayu Biswas ◽

Reazur Rahman ◽

Varun Gupta ◽

Michael Rosbash ◽

Robert H. Singer

Keyword(s):

Rna Editing ◽

False Positive ◽

Catalytic Domain ◽

False Negative ◽

Rna Regulation ◽

Rna Targets ◽

Bona Fide ◽

Actin Mrna ◽

Nascent Transcripts

AbstractNearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking (“CLIP” and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP (“TRIBE”). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β-actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β-actin gene. This result indicates that MCP-ADAR bound to β-actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.

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Variable In Vivo Hepatitis D Virus (HDV) RNA Editing Rates According to the HDV Genotype

Viruses ◽

10.3390/v13081572 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1572

Author(s):

Samira Dziri ◽

Christophe Rodriguez ◽

Athenaïs Gerber ◽

Ségolène Brichler ◽

Chakib Alloui ◽

...

Keyword(s):

Rna Editing ◽

Stop Codon ◽

Genome Replication ◽

Hepatitis D ◽

Reading Frame ◽

Delta Antigen ◽

B Virus ◽

Virion Morphogenesis ◽

Kinetics Of

Human hepatitis delta virus (HDV) is a small defective RNA satellite virus that requires hepatitis B virus (HBV) envelope proteins to form its own virions. The HDV genome possesses a single coding open reading frame (ORF), located on a replicative intermediate, the antigenome, encoding the small (s) and the large (L) isoforms of the delta antigen (s-HDAg and L-HDAg). The latter is produced following an editing process, changing the amber/stop codon on the s-HDAg-ORF into a tryptophan codon, allowing L-HDAg synthesis by the addition of 19 (or 20) C-terminal amino acids. The two delta proteins play different roles in the viral cell cycle: s-HDAg activates genome replication, while L-HDAg blocks replication and favors virion morphogenesis and propagation. L-HDAg has also been involved in HDV pathogenicity. Understanding the kinetics of viral editing rates in vivo is key to unravel the biology of the virus and understand its spread and natural history. We developed and validated a new assay based on next-generation sequencing and aimed at quantifying HDV RNA editing in plasma. We analyzed plasma samples from 219 patients infected with different HDV genotypes and showed that HDV editing capacity strongly depends on the genotype of the strain.

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Efficient precise in vivo base editing in adult dystrophic mice

10.1101/2020.06.24.169292 ◽

2020 ◽

Author(s):

Li Xu ◽

Chen Zhang ◽

Haiwen Li ◽

Peipei Wang ◽

Yandi Gao ◽

...

Keyword(s):

Rna Editing ◽

Deep Sequencing ◽

High Efficiency ◽

Contractile Function ◽

Functional Improvement ◽

Base Editing ◽

Monogenic Diseases ◽

Editing Activity ◽

Target Rna

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

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Fluoxetine improves behavioural deficits induced by chronic alcohol addiction by alleviating RNA editing of 5-HT2C receptors in astrocytes

10.1101/751065 ◽

2019 ◽

Author(s):

Zexiong Li ◽

Shanshan Liang ◽

Shuai Li ◽

Beina Chen ◽

Manman Zhang ◽

...

Keyword(s):

Alcohol Consumption ◽

Rna Editing ◽

Alcohol Intake ◽

Motor Coordination ◽

Alcohol Addiction ◽

Chronic Alcohol ◽

Chronic Alcohol Consumption ◽

Adenosine Deaminases ◽

Underlying Pathology

AbstractThe alcoholism and major depressive disorder are common comorbidity, with alcohol-induced depressive symptoms being eased by selective serotonin re-uptake inhibitors (SSRIs), although the mechanisms underlying pathology and therapy are poorly understood. Chronic alcohol consumption affects the activity of serotonin 2C receptors (5-HT2CR) by regulating adenosine deaminases acting on RNA (ADARs) in neurones. Astrogliopathic changes contribute to alcohol addiction, while decreased release of ATP from astrocytes can trigger depressive-like behaviours in mice. In this study, we discovered that chronic alcohol addiction increased editing of RNA of 5-HT2CR via up-regulating the expression of ADAR2, consequnetly reducing the release of ATP from astrocytes induced by 5-HT2CR agonist, MK212. At the same time SSRI antidepressant fluoxetine decreased the expression of ADAR2 through the transactivation of EGFR/PI3K/AKT/cFos signalling pathway. Reduction in ADAR2 activity eliminated the RNA editing of 5-HT2CR in vivo and increased release of astroglial ATP which was suppressed by chronic alcohol consumption. Meanwhile, fluoxetine improved the behavioural and motor symptoms induced by alcohol addiction and decreased the alcohol intake. Our study suggests that the astrocytic 5-HT2CR contribute to alcohol addiction; fluoxetine thus can be used to alleviate depression, treat alcohol addiction and improve motor coordination.

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