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Evidence of RNA editing in pig longissimus dorsi muscle
RNA editing refers to post-transcriptional modifications of an RNA transcript (excluding processes such as splicing or polyadenylation) that alter its sequence such that it no longer reflects the corresponding DNA sequence. In swine, as in all mammals, most RNA editing is thought to be catalyzed by adenosine deaminase acting on RNA, which substitutes adenosine for inosine in a site-specific and tissue-dependent manner. This is observed as an adenosine to guanine edit of RNA with respect to DNA in next-gen sequencing, where inosine is converted into guanine during cDNA library preparation. Our objective was to use next-gen sequencing data to identify potential RNA editing sites in pigs. We used genomic DNA and longissimus dorsi (LD) muscle total RNA from an F2 pig from the Michigan State University Duroc-Pietrain resource population. Non-strand specific polyA RNAseq data from LD was used to call candidate RNA editing sites. Whole genome sequence was used to call genotypes. DNAseq and RNAseq sequence reads were aligned to SScrofa 10.2 using bowtie2 and tophat, respectively. To minimize mapping error, only high quality bases (Phred Score>25) from uniquely mapped reads were used in the analysis. Candidate editing sites were required to have homozygous genotypes in DNAseq data, with a sequencing depth of at least 10 reads, with 95% or more of the reads in agreement. Five or more RNAseq reads with an alternative allele were required to give sufficient evidence for a possible editing event. A total of 686 candidate RNA editing sites were observed with A to G edits being the most common. These sites were found across all autosomes and the X chromosome, with the majority located in upstream and downstream gene proximal regions, intronic variants and UTRs. Since this analysis was done using a non-strand specific protocol, A to G and T to C sites cannot be distinguished a priori; the combined frequency of A to G and T to C sites observed was 38.34%. Results of this initial study indicate the overall composition of transcriptome-wide RNA editing in swine resembles other studies in primates, where A to G edit sites are the most common. In primates, RNA editing levels have been associated with diseases such as ALS, tumors and depression. Future efforts are warranted to identify RNA editing events in additional pig tissues, as well as determine the impacts of RNA editing on transcriptional regulation and economically important phenotypes.
Keywords: RNA editing, ADAR editing, transcriptional regulation