Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE168234 | GEO | 2021/03/05
REPOSITORIES: GEO
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Project description:We used CRISPR KI technology to mutagenize the cis-regulatory elements including the editing stem and the editing complementary sequence of three RNA editing substrates-NEIL1, TTYH2, AJUBA. We designed single nucleotide variants, double-nucleotide variants and other mutant isoforms to interrogate the primary sequence and the secondary structures around the endogenous RNA editing sites. Gene specific primers were used to make the NGS library for each locus. The unique mutation of each tested isoform can be used as barcode and RNA editing level can be measured for each isoform from the pooled library. We found that RNA sequence and structure features synergistically determine the editing levels. Several features, such as mutation number, free energy, and probability of active conformation, play an important role in determining editing efficiency. This study systematically investigated the contribution of cis-regulatory elements to ADAR1 RNA editing by combining CRISPR-based saturation mutagenesis and deep sequencing technology.
2020-06-01 | GSE138860 | GEO
Project description:Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.
2018-05-31 | GSE99789 | GEO
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
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.
2013-06-17 | E-GEOD-47997 | biostudies-arrayexpress
Project description:Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase, initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA-Seq screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3′ untranslated regions (3′ UTRs). Further analysis revealed several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The identification of multiple mRNA editing substrates for APOBEC1 suggests additional functions for this cytidine deaminase beyond its characterized role in lipid absorption.
2011-01-23 | GSE24958 | GEO
Project description:RNA base editing represents a promising alternative for genome editing. Recent approaches harness the endogenous RNA editing enzyme ADAR to circumvent problems related to the ectopic expression of an editing enzyme, but they suffer from sequence restriction, lack of efficiency, and bystander editing. Here, we present in‐silico optimized CLUSTER guide RNAs, which bind their target mRNAs in a multivalent fashion and thereby enable editing with unprecedented precision as shown by next generation sequencing. CLUSTER guide RNAs can be genetically encoded and manufactured into viruses to work in various cell lines. They achieve on‐target editing on endogenous transcripts like GUSB and NUP43 with yields up to 45% without bystander editing and have been shown to recruit endogenous ADAR in vivo. The CLUSTER approach tremendously enlarges the sequence space available for guide RNA design and opens new avenues for drug development in the field of RNA base editing.
2021-09-20 | GSE184244 | GEO
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
Project description:A-to-I RNA editing levels differ across tissues and cell types, but regulators of the editing process are largely unknown. We performed RNA-seq on M17 and HeLa cells with overexpression of different candidate Adar regulators and GFP as a control to assay editing level differences between overexpression and control.
2019-05-07 | GSE130770 | GEO
Project description:Endogenous double-stranded RNA is prevented from activating the cytosolic antiviral receptor MDA5 by adenosine-to-inosine editing by ADAR. Consequently, CRISPR/cas9 targeting ADAR in the human monocytic cell line THP-1 induces a spontaneous MDA5-dependent interferon response. This response is synergistically enhanced by concomitant CRISPR/cas9 targeting of hnRNPC. For this study, cas9 transgenic THP-1 were nucleofected with combinations of control gRNA, ADAR gRNA or hnRNPC gRNA to elucidate the basis for synergistic interferon-stmulated gene induction by combined ADAR and hnRNPC targeting. As a control for interferon-stimulated gene induction, IFN-alpha treated THP-1 were included, as well. To investigate potential interferon inducing mechanisms, we quantified gene expression, intron expression as well as the expression of regions rich in adenosine-to-inosine editing clusters. In a second part, nuclear vs cytosolic distribution of RNA rich in adenosine-to-inosine clusters was investigated by sequencing of total RNA or RNA from cytosolic and nuclear extracts of ADAR and/or hnRNPC targeted cells. In the first part of the study, STING-deficient, cas9 transgenic THP-1 were nucleofected with combinations of control, GFP-targeting, ADAR-targeting or hnRNPC-targeting gRNA and cells collected for total RNA extraction on days three, four and five after nucleofection. In addition, another control-nucleofected sample was treated on day 3 for 24 h. The experiment was repeated three times leading to three replicates per condition. In the second part, WT cas9-transgenic THP-1 were nucleofected with combinations of control, ADAR-targeting or hnRNPC-targeting gRNA and cells were either collected for total RNA extraction or separated into digitonin-soluble (cytosolic) and digitonin-resistant (nuclear and mitochondrial) compartments and then RNA extracted. The experiment was repeated twice leading to two replicates per condition. Differential gene expression analysis confirmed synergistic induction of interferon-stimulated genes. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters or regions enriched in editing clusters (ECRs) in general. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. Part 2 of the study confirmed cytosolic access of a subset of ECRs to the cytosol.
2021-07-16 | GSE176012 | GEO
Project description:Purpose: The purpose of this experiment is to expand the repertoire of C. elegans edited transcripts and identify the roles of ADR-1 as indirect regulator of editing and ADR-2 as the only active deaminase in vivo. Methods: Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline. Results: Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3’ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation. Conclusions: ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms. Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline.
2014-01-20 | E-GEOD-51556 | biostudies-arrayexpress