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

2019 ◽  
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.

scholarly journals Site-directed RNA repair of endogenous Mecp2 RNA in neurons

2017 ◽  
Vol 114 (44) ◽  
pp. E9395-E9402 ◽  
Author(s):  
John R. Sinnamon ◽  
Susan Y. Kim ◽  
Glen M. Corson ◽  
Zhen Song ◽  
Hiroyuki Nakai ◽  
...  
Keyword(s):  
Rna Editing ◽  
Protein Function ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Mrna Level ◽  
Gene Encoding ◽  
Rna Repair ◽  
Mecp2 Protein ◽  
Localization Signal

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

scholarly journals Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

2021 ◽  
Author(s):  
Wessel van Leeuwen ◽  
Michael VanInsberghe ◽  
Nico Battich ◽  
Fredrik Salmen ◽  
Alexander van Oudenaarden ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Rna Binding Proteins ◽  
Single Cells ◽  
Stress Granules ◽  
Stress Granule ◽  
Rna Content ◽  
Editing Enzyme

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

scholarly journals APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo

2021 ◽  
Author(s):  
Saeed Soleymanjahi ◽  
Valerie Blanc ◽  
Nicholas Davidson
Keyword(s):  
Rna Editing ◽  
Nearest Neighbor ◽  
Cytidine Deaminase ◽  
Target Sequence ◽  
Flanking Sequence ◽  
Cis Acting ◽  
Rna Targets ◽  
Genome Wide ◽  
Nucleotide Modification

Mammalian C-to-U RNA editing was described more than 30 years ago as a single nucleotide modification in APOB RNA in small intestine, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. Co-factor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF observed to be edited at a lower frequency than RBM47 dominant targets. The model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.

scholarly journals DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes

2020 ◽  
Vol 48 (20) ◽  
pp. e117-e117
Author(s):  
Kun Tian ◽  
Yongjian Guo ◽  
Bingjie Zou ◽  
Liang Wang ◽  
Yun Zhang ◽  
...  
Keyword(s):  
Rna Editing ◽  
Human Cells ◽  
Hairpin Dna ◽  
Flap Endonuclease 1 ◽  
Dna And Rna ◽  
Rna Targets ◽  
Single Base Mismatch ◽  
Compact Size

Abstract Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

scholarly journals Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

2017 ◽  
Author(s):  
Weijin Xu ◽  
Reazur Rahman ◽  
Michael Rosbash
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Target Identification ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Targets ◽  
Rna Targets ◽  
Editing Activity ◽  
Editing Enzyme

AbstractWe previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR and performs Adenosine-to-Inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

scholarly journals In vivo repair of a protein underlying a neurological disorder by programmable RNA editing

2020 ◽  
Author(s):  
John R Sinnamon ◽  
Susan Y Kim ◽  
Jenna R Fisk ◽  
Zhen Song ◽  
Hiroyuki Nakai ◽  
...  
Keyword(s):  
Mouse Model ◽  
Rna Editing ◽  
Neurological Disease ◽  
Catalytic Domain ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Neurodevelopmental Disease ◽  
Mecp2 Protein

AbstractRNA base editing is gaining momentum as an approach to repair mutations, but its application to neurological disease has not been established. We have succeeded in directed transcript editing of a pathological mutation in a mouse model of the neurodevelopmental disease, Rett syndrome. Specifically, we directed editing of a guanosine to adenosine mutation in RNA encoding Methyl CpG Binding Protein 2 (MECP2). Repair was mediated by injecting the hippocampus of juvenile Rett mice with an adeno-associated virus expressing both an engineered enzyme containing the catalytic domain of Adenosine Deaminase Acting on RNA 2 and a Mecp2 targeting guide. After one month, 50% of Mecp2 RNA was recoded in three different hippocampal neuronal subtypes, and the ability of MeCP2 protein to associate with heterochromatin was similarly restored to 50% of wild-type levels. This study represents the first in vivo programmable RNA editing applied to a model of neurological disease.

scholarly journals Geminivirus-encoded TrAP suppressor inhibits the histone methyltransferase SUVH4/KYP to counter host defense

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Claudia Castillo-González ◽  
Xiuying Liu ◽  
Changjun Huang ◽  
Changjiang Zhao ◽  
Zeyang Ma ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Systemic Infection ◽  
Histone Methyltransferase ◽  
Transcriptional Gene Silencing ◽  
Antiviral Defense ◽  
Dna Viruses ◽  
Cellular Target ◽  
Bona Fide

Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase

2018 ◽  
Vol 31 (12) ◽  
pp. 471-478 ◽  
Author(s):  
Sonali Bhakta ◽  
Md Thoufic Anam Azad ◽  
Toshifumi Tsukahara
Keyword(s):  
Genetic Code ◽  
Rna Editing ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Genetic Diseases ◽  
Site Directed Mutagenesis ◽  
Sequencing Analysis

Abstract Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5′ than for the 3′A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Sign in / Sign up

Export Citation Format

Share Document