Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

Abstract

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs.

A-to-I RNA editing independent of ADARs in filamentous fungi

“…When analyzed for the flanking sequences of the edited As, the ¡1 position has a strong preference for U in both F. graminearum 13 and F. verticillioides (Fig. 1A).…”

“…Genome-wide analysis with strand-specific RNA-seq data generated with RNA isolated from perithecia identified 26,056 A-to-I editing sites. 13 In RNA samples isolated from conidia (asexual spores) and vegetative hyphae, no A-to-I editing sites were identified. 13 The average editing level is 14.8% in F. graminearum, which is similar to that of animals.…”

“…13 In RNA samples isolated from conidia (asexual spores) and vegetative hyphae, no A-to-I editing sites were identified. 13 The average editing level is 14.8% in F. graminearum, which is similar to that of animals. 14,15 However, genes that are functionally related to sexual reproduction or up-regulated during sexual reproduction tend to have editing sites with higher editing levels in fungi.…”

“…Stage-specific RNA editing also occurs during sexual reproduction in F. verticillioides. 13 Analysis with published RNA-seq data 16,17 showed that genome-wide A-to-I RNA editing also occurs in the model filamentous fungus Neurospora crassa 13 and its closely related species N. tetrasperma. However, because these published RNA-seq data of F. verticillioides and Neurospora species were un-stranded and generated from early sequencing platforms with short single end reads, they had high error rate and were not suitable for systematic identification of A-to-I editing sites.…”

“…15,[18][19][20] In contrast, 70.5% of A-to-I editing sites in F. graminearum occur in the coding regions (CDSs) and over 2-thirds of them result in amino acid changes. 13 A total of 4,594 genes have at least one missense editing event during sexual reproduction. Because missense editing sites tend to have higher editing levels than synonymous editing sites and 76.2% of missense editing events cause changes to amino acids of different physicochemical properties, 13 it is likely that A-to-I editing is important for adaptation and diversification protein functions during sexual reproduction in F. graminearum.…”