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HyperTRIBE uncovers MEX3A RNA-binding activity in colorectal cancer (CRC) cells

ABSTRACT: A comprehensive list of RNA targets of the RNA-binding protein MEX3A remains elusive. To address this gap, we applied the high-throughput HyperTRIBE (Targets of RNA Identified By Editing) methodology in the SW480 cell line in order to map the MEX3A RNA interactome in CRC. An hyperactive version of the catalytic domain (cd) of the fruit fly RNA-editing enzyme Adenosine Deaminase Acting on RNA (ADAR) was fused either to the human MEX3A coding sequence (MEX3A-hyperADARcd) or its RNA-binding mutant version (MEX3ADKH-hyperADARcd) as a control. Once expressed, the MEX3A-hyperADARcd fusion protein creates irreversible adenosine-to-inosine (A-to-I) RNA editing events that are identified as adenosine-to-guanosine (A-to-G) mismatches near MEX3A-binding sites by RNA-sequencing. Editing specificity is guaranteed by the lack of RNA-binding of ADARcd. To prevent analytical bias by ADAR continuous activity, the MEX3A-hyperADARcd and MEX3ADKH-hyperADARcd sequences were placed under the control of a doxycycline (DOX)-inducible promoter for transient expression, together with a TurboRFP reporter for sorting of positive cells expressing the constructs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312175 | GEO | 2026/03/30

REPOSITORIES: GEO

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

Project description:Most current methods to identify cell-specific RNA binding protein (RBP) targets require analyzing an extract, a strategy that is problematic with small amounts of material. We previously addressed this issue by developing TRIBE, a method that expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR. TRIBE performs Adenosine-to-Inosine editing on candidate RNA targets of the RBP. However, target identification is limited by the 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 than TRIBE, many of which are also edited by TRIBE but at a much lower editing frequency. The data have mechanistic implications for the enhanced editing activity of the HyperADARcd as part of a RBP fusion protein and also indicate that HyperTRIBE more faithfully recapitulates the known binding specificity of its RBP than TRIBE.

2017-08-19 | GSE102814 | GEO

The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [hyperTRIBE]

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2023-07-26 | GSE224485 | GEO

HyperTRIBE analysis of the m6A reader YTHDF1 in HEK293T cells

Project description:TRIBE/HyperTRIBE has been developed as an alternative method for transcriptome-wide mapping of the RNA targets of RBPs. This method involves fusing an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR. When these fusion proteins are expressed in target cells, they direct A-to-I editing at residues in proximity to RNA binding sites. To demonstrate the utility of HyperTRIBE approach, we applied HyperTRIBE to the m6A reader YTHDF1 in HEK293T cells.

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

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Comparison of STAMP and TRIBE for identifying the targets of RNA-binding proteins in human and Drosophila cells [Drosophila]

Project description:RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to a RBP. In STAMP, the ADARcd is replaced by the RNA editing enzyme Apobec (REF). Here we compared the two enzymes fused to the RBP TDP43 in human cells. Although they both identified TDP43 target mRNAs, combining the two methods more successfully identified high confidence targets. We also assayed the two enzymes in Drosophila cells in which RBP-Apobec fusions generated only low numbers of editing sites comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor (Drosophila EIF4E-BP), and contrasted with successful RBP-ADARcd fusions. The results indicate that TRIBE is the method of choice in Drosophila.

2023-02-05 | GSE223555 | GEO

Comparison of STAMP and TRIBE for identifying the targets of RNA-binding proteins in human and Drosophila cells [human]

2023-02-05 | GSE223556 | GEO

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Project description:Adenosine deaminases, RNA specific (ADAR) are proteins that deaminate adenosine to inosine which is then recognized in translation as guanosine. To study the roles of ADAR proteins in RNA editing and gene regulation, we carried out DNA and RNA sequencing, RNA interference and RNA-immunoprecipitation in human B-cells. We also characterized the ADAR protein complex by mass spectrometry. The results uncovered over 60,000 sites where the adenosines (A) are edited to guanosine (G) and several thousand genes whose expression levels are influenced by ADAR. We also identified more than 100 proteins in the ADAR protein complex; these include splicing factors, heterogeneous ribonucleoproteins and several members of the dynactin protein family. Our findings show that in human B-cells, ADAR proteins are involved in two independent functions: A-to-G editing and gene expression regulation. In addition, we showed that other types of RNA-DNA sequence differences are not mediated by ADAR proteins, and thus there are co- or post-transcriptional mechanisms yet to be determined. Here we studied human B-cells where ADAR proteins (ADAR1 and ADAR2) are expressed but APOBECs are not. We identified the sequence differences between DNA and the corresponding RNA in B-cells from two individuals. Then, we carried out RNA interference, RNA-immunoprecipitation and next generation sequencing to determine the contribution of ADAR proteins in mediating A-to-G editing and other types of RNA-DNA sequence differences.

2013-09-20 | E-GEOD-38233 | biostudies-arrayexpress

RNA Editing in breast cancer

Project description:High-throughput sequencing screens suggest that RNA editing, which consists in the substitution of adenosine with inosine by the RNA-specific adenosine deaminase (ADAR) enzyme, occurs at several thousand positions across the human genome. Recent evidences have shown that RNA-editing could promote proliferation and carcinogenesis; however, the general principles of ADAR activity on the transcriptome and how ADAR is controlled in cancers remain to be established. The main aim of this project was to investigate the phenomenon of RNA editing in breast and other cancers. The frequency of A-to-I editing was evaluated in 58 breast cancers equally distributed among the different molecular subtypes and 10 normal breast tissues. The analysis was focused on defining: the relationship between the global amount of editing and ADAR expression; the ability to predict the level of editability of specific sites; the distribution of editing in normal and tumour samples and among different breast cancer subtypes; and the clinical, pathological and genomic factors affecting editing.

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HyperTRIBE analysis of the m6A readers in hESCs

Project description:TRIBE/HyperTRIBE has been developed as an alternative method for transcriptome-wide mapping of the RNA targets of RBPs. This method involves fusing an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR. When these fusion proteins are expressed in target cells, they direct A-to-I editing at residues in proximity to RNA binding sites. The control plasmid, mammalian expression plasmid, containing mCherry ADAR control and p2A GFP reporter can be obtained from Addgene (Plasmid #154786). As for RBP-TRIBE plasmid, using standard restriction enzyme-based cloning to clone RBP (DDX6, FUBP3, FXR2 or L1TD1) into TRIBE plasmid in hESCs.

2026-02-09 | GSE288304 | GEO

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [expression]

Project description:Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.

2013-06-15 | E-GEOD-47945 | biostudies-arrayexpress

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