Dataset Information

High conservation of translationally significant RNA editing sites in hyper-editing ferns implies they are not selectively neutral

ABSTRACT: RNA editing is essential for processing transcripts in plant chloroplasts and mitochondria. Levels of RNA editing vary between lineages, but some hornworts, lycophytes and ferns have an extraordinary abundance of RNA editing. A feature of ‘hyper-editing’ species is the prevalence of RNA editing events which influence translation by affecting start codons and stop codons via C-to-U editing and U-to-C editing respectively. These ‘translationally significant’ RNA editing events may play roles in regulating organelle gene expression. To investigate the importance of translationally significant RNA editing events, we generated DNA and RNA sequence libraries for four Salviniales water ferns: Azolla rubra, Azolla pinnata, Marsilea mutica and Salvinia molesta. We assembled chloroplast genomes, mitochondrial genes and transcriptomes, and identified RNA editing sites and candidate RNA editing factors. We re-analysed sequence data of Azolla filiculoides and conducted a comparative analysis of RNA editing in chloroplasts and mitochondrial transcripts. Estimates of pyrimidine transition rates show that translationally significant RNA editing sites are more conserved than other non-synonymous editing sites, suggesting an emergent role in organelle gene expression that is not selectively neutral. Shotgun proteomics of Marsilea mutica chloroplast thylakoid fractions verified the expected consequences of RNA editing on translation of chloroplast transcripts and implies that mechanisms exist to avoid translation of partially edited transcripts. Start codon editing may be one of those mechanisms.

INSTRUMENT(S):

ORGANISM(S): Marsilea Mutica

TISSUE(S): Frond

SUBMITTER: Owen Duncan

LAB HEAD: Ian Small

PROVIDER: PXD065205 | Pride | 2025-10-27

REPOSITORIES: Pride

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Publications

High Conservation of Translation-Enabling RNA Editing Sites in Hyper-editing Ferns Implies They Are Not Selectively Neutral.

Kwok van der Giezen Farley M FM McDowell Rose R Duncan Owen O Zumkeller Simon S Colas des Francs-Small Catherine C Small Ian I

Molecular biology and evolution 20251001 10

RNA editing is essential for processing transcripts in plant chloroplasts and mitochondria. Levels of RNA editing vary between lineages, but some hornworts, lycophytes, and ferns have an extraordinary abundance of RNA editing. A feature of "hyper-editing" species is the prevalence of RNA editing events that promote translation by creating start codons or removing stop codons via C-to-U or U-to-C editing, respectively. These "translation-enabling" RNA editing events may play roles in regulating o ...[more]

PMID: 41024750

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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.

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	CathyA-F01.mzML	Mzml
	CathyA-F02.mzML	Mzml
	CathyA-F03.mzML	Mzml
	CathyA-F04.mzML	Mzml
	CathyA-F05.mzML	Mzml