Unknown,Transcriptomics,Genomics,Proteomics

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Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila

ABSTRACT: The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila, by isolating nascent RNA from adult fly heads and subjecting samples to high-throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR null mutant strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally. GSM914095: Fly genomic DNA sequencing. Sequenced on the Illumina GA II. GSM914102-GSM914113: Fly head nascent RNA profiles over 6 time points of a 12hr light:dark cycle in duplicate; sequenced on the Illumina GA II. GSM914114-GSM914119: Fly head nascent RNA profiles of yw, FM7, ADAR0 males in duplicate; sequenced on the HiSeq2000. GSM915213-GSM915214: Fly head mRNA profiles over 2 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II. GSM915215-GSM915220: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; paired-end sequenced on the Illumina GA II. GSM915221-GSM91526: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Joseph Rodriguez

PROVIDER: E-GEOD-37232 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila.

Rodriguez Joseph J Menet Jerome S JS Rosbash Michael M

Molecular cell 20120531 1

The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila by isolating nascent RNA from adult fly heads and subjecting samples to high throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR-null strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels co ...[more]

PMID: 22658416

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Project description:Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) RNA editing sites in wild type mice that were not edited or reduced in editing frequency in ADAR1 deficient murine erythroid cells. Secondly, to determine the transcription consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Total RNA from E14.5 fetal liver of embryos with an erythroid restricted deletion of ADAR1 (KO) and littermate controls (WT), in duplicate. cDNA libraries were prepared and RNA sequenced using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRTâPCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using BurrowsâWheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.7, p<0.0001 between biological duplicates per genotype. Clusters of hyper-editing were onserved in long, unannotated 3'UTRs of erythroid specific transcripts. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 5 log2FC) in KO fetal liver compared to WT. 11.332 (6,894 novel) A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data. Conclusions: Our study represents the first detailed analysis of erythroid transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to prevent sensing of endogenous transcripts, likely via a RIG-I like receptor mediated axis. Fetal liver mRNA profiles of E14.5 wild type (WT) and ADAR Epor-Cre knock out mice were generated by deep sequencing, in duplicate using Illumina HiSeq 2000.

Project description:Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) mismatches in RNA-seq data compared to mm9 reference genome in wild type mice that were not edited or reduced in editing frequency in ADAR1E861A editing deficient mice. Secondly, to determine the transcriptional consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Fetal liver mRNA profiles of embryonic day 12.5 wild-type (WT) and ADAR1 editing-deficient (ADAR1E861A) mice were generated by RNA sequencing, in triplicate (biological replicates), using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.94 between biological triplicates per genotype. Approximately 4.4% of the transcripts showed differential expression between the WT and ADAR1E861A fetal liver, with a LogFC≥1.5 and p value <0.05. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 11 logFC) in ADAR1E861A fetal liver compared to WT. 6,012 A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data of WT and ADAR1E861A fetal liver. Conclusions: Our study represents the first detailed analysis of fetal liver transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to suppress interferon signaling to endogenous RNA. Fetal liver mRNA profiles of E12.5 wild type (WT) and ADAR E861A mutant mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 200.

Dataset Information

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila

Publications

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila.

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