Lessons from discovery of true ADAR RNA editing sites in a human cell line
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ABSTRACT:
PROVIDER: PRJNA918405 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22975000_1.fastq.gz | Fastqsanger.gz | |||
| SRR22975000_2.fastq.gz | Fastqsanger.gz | |||
| SRR22975001.fastq.gz | Fastqsanger.gz | |||
| SRR22975001_1.fastq.gz | Fastqsanger.gz | |||
| SRR22975001_2.fastq.gz | Fastqsanger.gz |
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Project description:Conversion, or editing, of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common naturally occurring post-transcriptional RNA modifications found in multiple species from fruit flies to humans with emerging connection to disease, particularly cancer. The A-to-I conversion can happen at specific sites in certain RNA molecules and lead to mRNA recoding and increase in proteome diversity, changes in RNA stability, splicing and regulation. Such sites could be detected as adenine-to-guanine sequence changes by RNA sequencing. Thus, not surprisingly, the advent of next-generation sequencing has caused a massive explosion of reported sites detected by a slew of genome-wide studies in different species leading to wide ranges of conclusions about biological implications of the A-to-I editing on the physiology of normal and cancerous cells. Nonetheless, extensive independent validation of the authenticity of the reported sites is often missing in such endeavors which combined with the relatively high error rate of next generation sequence leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. Therefore, in this work, we provide a comprehensive and an extensively validated list of A-to-I editing sites in a human cancer cell line. Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases can not be validated. We provide a potential solution to these problems and discuss their ramifications to the current state of genomics of RNA editing as well as potential biological conclusions drawn from analysis of the compendium of true A-to-I editing sites in a human cancer cell line.
2023-06-05 | GSE222170 | GEO
Project description:BackgroundConversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them.ResultsStrikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification.ConclusionsAccurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.
| S-EPMC10357658 | biostudies-literature
Project description:Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish. To assess the effect of Adar KD and OE, uninjected wild-type and embryos injected with 1 ng of adar MO or 50 pg of adar mRNA were kept until desired developmental stage: 128-cell or 50% epiboly. Two replicates of 20 pooled embryos from each time point were isolated for RNA extraction. For the analysis at 12 hpf, a separate batch of uninjected control, as well as Adar KD and OE samples were generated in three replicates.
2022-08-02 | GSE182714 | GEO
Project description:ADAR (adenosine deaminase that acts on RNA) editing enzymes target coding and noncoding double-stranded RNA (dsRNA) and are essential for neuronal function. Early studies showed that ADARs preferentially target adenosines with certain 5' and 3' neighbours. Here we use current Sanger sequencing protocols to perform a more accurate and quantitative analysis. We quantified editing sites in an ∼800-bp dsRNA after reaction with human ADAR1 or ADAR2, or their catalytic domains alone. These large data sets revealed that neighbour preferences are mostly dictated by the catalytic domain, but ADAR2's dsRNA-binding motifs contribute to 3' neighbour preferences. For all proteins, the 5' nearest neighbour was most influential, but adjacent bases also affected editing site choice. We developed algorithms to predict editing sites in dsRNA of any sequence, and provide a web-based application. The predictive power of the algorithm on fully base-paired dsRNA, compared with biological substrates containing mismatches, bulges and loops, elucidates structural contributions to editing specificity.
| S-EPMC3113232 | biostudies-literature
Project description:RNA editing can be a promising therapeutic approach. However, ectopic expression of RNA editing enzymes was found to trigger off-target editing, and the recruitment of endogenous adenosine deaminase acting on RNA (ADAR) suffers from low efficiency and fluctuating ADAR expression. Here, we identified ADAR inhibitors (ADIs) that suppressed the activity of the fused ADAR2 deaminase domain (ADAR2DD). Using ADI, we developed an RNA transformer adenine base editor (RtABE) with both high specificity and high efficiency. With the fusion of ADI to ADAR2DD, RtABE remains inactive until it binds its target site. After binding to the target site, ADI is cleaved from ADAR2DD and RtABE becomes active. RtABE induced efficient on-target editing in various cells with different ADAR expression levels. Delivering RtABE via an adeno-associated virus enabled up to a 45% RNA correction rate in Hurler syndrome mice with no significant off-target editing, and -L-iduronidase activity was restored. RtABE is a highly specific and efficient RNA editing system with broad applicability.
2025-02-06 | GSE267726 | GEO
Project description:True mangrove rhizospheres Raw sequence reads
| PRJNA826370 | ENA
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: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:Gene regulation by small RNAs and ADAR RNA editing
| PRJNA353652 | ENA
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