Abstract
A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.