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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

ABSTRACT: Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

PROVIDER: PRJNA666136 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12729451_1.fastq.gz	Fastqsanger.gz
	SRR12729451_2.fastq.gz	Fastqsanger.gz
	SRR12729452_1.fastq.gz	Fastqsanger.gz
	SRR12729452_2.fastq.gz	Fastqsanger.gz
	SRR12729453_1.fastq.gz	Fastqsanger.gz

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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Project description:We use RNA-seq to study the transcriptome wide specificity profiles of A-to-I RNA editing by split-ADAR2 constructs.

2022-01-19 | GSE158656 | GEO

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.

Project description:Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5' guanosine is very low thus limiting their utility as a transcriptome engineering toolset. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.

| S-EPMC8809894 | biostudies-literature

Next Generation Sequencing Analysis of Wild Type, ADAR2 over expressing and ADAR2+SRSF9 overexpresssing SH-Sy5y cells Transcriptomes

Project description:Purpose: A-to-I RNA editing is critical for many cellular processes. The sites of A-I RNA editing can be identified through RNA-seq and matching the reads to the annotated database like RADAR. The goals of this study is to compare A-I RNA editing profile among wild type, ADAR2 overexpressing and ADAR2+SRSF9 overexpressing 293T cells to evaluate the influence of SRSF9 repressive action on A-I RNA editing Methods: A-I RNA editing profile and gene expression profiles were generated by deep sequencing from all the samples, using NEBNext® Ultra™ Directional RNA Library Prep Kit. The sequence reads that passed quality filters were analyzed for identifying the A-I RNA editing sites using RADAR Database Results: Upon overexpression of ADAR2, we found that 18,174 sites were differentially edited compared to control cells, of which 97.0% showed a significant increase in their editing levels as expected (P < 0.05, Fisher’s test). Furthermore, when we overexpressed both ADAR2 and SRSF9 together, we found that 6,994 sites were differentially edited compared to overexpression of the deaminase alone (P < 0.05, Fisher’s test). Importantly, the editing of 92.5% of these sites was down-regulated, consistent with the function of SRSF9 as a repressor of editing. Conclusions: Our study reports a detailed analysis of the effect of SRSF9 on A-I RNA editing of ADAR2-specific targets. We Conclude that this SRSF9 regulon is significantly enriched for brain-specific editing sites despite our unbiased approach.

2018-06-21 | GSE114970 | GEO

Next Generation Sequencing Analysis of Wild Type, ADAR2 over expressing and ADAR2+SRSF9 overexpresssing HEK 293T cells Transcriptomes

2018-06-21 | GSE108126 | GEO

MiRNAinU118_Cells_Inactive_vs_ active_ Adar2

Project description:MicroRNA expression profile of NB U118 cells transfected with a plasmid directing an inactive Adar2 vs an active Adar2 - 3 biological replicates 1 Condition experiment, 3 biological replicates, 2 replicates/array

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MiRNAinU118_Cells_Inactive_vs_ active_ Adar2

Project description:MicroRNA expression profile of NB U118 cells transfected with a plasmid directing an inactive Adar2 vs an active Adar2 - 3 biological replicates

2015-02-28 | GSE63694 | GEO

Changes in gene expression and RNA editing in Adar2-WT and Adar2-KO Mouse embryonic Fibroblasts (MEFs)

Project description:Purpose: Measure gene expression and RNA editing changes in Adar-WT and Adar2-KO MEFs Methods: Performed PolyA+ RNA-seq (single end, one sample each) of Adar2-WT and Adar2-KO MEFs Results: Observed changes in editing and gene expression of several genes Conclusion: Adar2 influences gene expression and editing of transcripts

2016-12-23 | GSE74746 | GEO

ADAR2 in HEK293T cells

Project description:ADAR2 in HEK293T cells

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Expression data from human oligodendroglia cells expressing shRNA targeting ADAR2 or control.

Project description:To assess whether antiviral genes are upregulated by ADAR2 knockdown, we performed a comprehensive analysis using a microarray system for oligodendroglia cells introduced shRNA against ADAR2 or control. The inflammation- and immune response-related genes were upregulated in ADAR2-knockdown cells.

2019-10-17 | GSE138927 | GEO

ADAR2 affects mRNA coding sequence edits but not gene expression or splicing in vivo

Project description:Adenosine deaminases (ADARs) are RNA binding proteins that bind to double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, including AMPA-subtype Glutamate channels such as Gria2 that is edited to introduce a Q to R amino acid change. Adar2 knock out mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on the frontal cortices of Adar2-/- Gria2R/R mice and their Adar2+/+ Gria2R/R littermates, quantifying overall gene expression, splicing, and A to I editing in a transcriptome-wide fashion. We found 56 editing sites whose level of editing was significantly diminished in the Adar2 deficient animals. The majority of Adar2 responsive editing sites were in the coding regions of genes. Interestingly, other than Adar2 expression, there were only two additional statistically significant differentially expressed genes, Flnb and Cdh13. There were also only three exons that showed statistically significant differences in expression levels between the two genotypes. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has only modest effects on gene expression or splicing in vivo. 6 ADAR2 Knock out frontal cortices and 6 litter mate control frontal cortices from male mice

2016-07-03 | E-GEOD-70588 | biostudies-arrayexpress

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