<p>A-to-I mRNA editing is an important co- or post-transcriptional event that can recode the heredity information through adenosine deaminase. It is mediated by <u>a</u>denosine <u>d</u>e<u>a</u>minases <u>a</u>cting on <u>R</u>NA (ADAR) in animals. Due to the lack of ADAR orthologs, yeast and filamentous fungi are assumed to have no A-to-I mRNA editing. However, genome-wide A-to-I mRNA editing was discovered in the plant pathogenic fungus <i>Fusarium graminearum</i> that occurs specifically during sexual reproduction in 2016. In a previous study all the predicted adenosine/cytosine deaminase genes except <i>FgTAD2 </i>and <i>FgTAD3</i>, the orthologs of yeast <i>TAD2 </i>and <i>TAD3</i>, were found to be dispensable for A-to-I mRNA editing in <i>F. graminearum</i>. <i>TAD2</i> and <i>TAD3</i> encode ADATs (<u>a</u>denosine <u>d</u>e<u>a</u>minase acting on <u>t</u>RNA) that mediate A-to-I editing at A34 of tRNA. In this study, <i>FgTAD2</i> was found to have two isoforms based on RNA-seq analysis. Whereas the longer isoform was predominant during vegetative growth, the expression of the short one was significantly increase and likely acts as the major isoform during sexual stage. Because deletion of <i>FgTAD2</i> appeared to be lethal, the RIP (<u>r</u>epeat-<u>i</u>nduced <u>p</u>oint mutation) approach was used to generate point mutations. A total of 16 RIP mutations were identified in <i>FgTAD2</i>after sequencing analysis with 8 ascospore progeny that were normal in vegetative growth but defective in sexual reproduction. Two of them were verified by introducing specific point mutations into the endogenous <i>FgTAD2</i> allele in the wild-type strain. In addition to genetic approaches, we developed an <i>in vitro</i> assay to detect the deaminase activity of FgTad2. The FgTad2 protein complex purified from perithecia formed by transformants of <i>F. graminearum</i> expressing FgTad2-6xHis by immunoprecipitation was found to catalyze A-to-I editing in a mRNA substrate. Like yeast Tad3, FgTad3 has the E to V mutation in its catalytic core that likely abolishes its ADAT activity but it forms heterodimers with FgTad2 based on co-immunoprecipitation assays. Because <i>FgTAD2</i> and <i>FgTAD3</i> were constitutively expressed and the FgTad2/FgTad3 protein complex purified from vegetative hyphae had no A-to-I RNA editing activity in <i>in vitro</i> assays, it is likely that stage-specific co-factors present in perithecia interact with FgTad2/FgTad3 ADATs (lack of RNA binding domains) and enable the editing of mRNA. Affinity purification and mass spectrometry were conducted with the FgTad2-S-tag and FgTad3-S-tag transformants. Among the putative FgTad2- and FgTad3-interacting proteins, Gad1 was confirmed to interact with FgTad2 specifically during sexual reproduction. Surprisingly, both the number of editing sites and editing levels were increased in the <i>gad1</i> mutant, indicating that Gad1 affects A-to-I mRNA editing in a negative way. Overall, genetic studies and <i>in vitro</i> assays showed that FgTad2, possibly together with FgTad3, is responsible for A-to-I mRNA editing. Proteins co-immunoprecipitated with FgTad2 likely contains co-factors interacting with FgTad2/FgTad3 for stage-specific A-to-I mRNA by these two ADATs in <i>F. graminearum</i>. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/17148056 |
| Date | 19 December 2021 |
| Creators | Zhuyun Bian (11797241) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Stage-specific_A-to-I_mRNA_editing_is_mediated_by_FgTad2_and_regulated_together_with_co-factors_in_Fusarium_graminearum/17148056 |
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