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ABSTRACT Adenosine-to-inosine (A-to-I) RNA editing independent of adenosine deaminase acting on RNA (ADAR) enzymes was discovered in fungi recently, and shown to be crucial for sexual reproduction. However, the underlying mechanism for editing is unknown. Here, we combine genome-wide comparisons, proof-of-concept experiments, and machine learning to decipher cis -regulatory elements of A-to-I editing in Fusarium graminearum . We identified plenty of RNA primary sequences and secondary structural features that affect editing specificity and efficiency. Although hairpin loop structures contribute importantly to editing, unlike in animals, the primary sequences have more profound influences on editing than secondary structures. Nucleotide preferences at adjacent positions of editing sites are the most important features, especially preferences at the −1 position. Unexpectedly, besides the number of positions with preferred nucleotides, the combination of preferred nucleotides with depleted ones at different positions are also important for editing. Some cis -sequence features have distinct importance for editing specificity and efficiency. Machine learning models built from diverse sequence and secondary structural features can accurately predict genome-wide editing sites but not editing levels, indicating that the cis -regulatory principle of editing efficiency is more complex than that of editing specificity. Nevertheless, our model interpretation provides insights into the quantitative contribution of each feature to the prediction of both editing sites and levels. We found that efficient editing of FG3G34330 transcripts depended on the full-length RNA molecule, suggesting that additional RNA structural elements may also contribute to editing efficiency. Our work uncovers multidimensional cis -regulatory elements important for A-to-I RNA editing in F. graminearum , helping to elucidate the fungal editing mechanism. IMPORTANCE A-to-I RNA editing is a new epigenetic phenomenon that is crucial for sexual reproduction in fungi. Deciphering cis -regulatory elements of A-to-I RNA editing can help us elucidate the editing mechanism and develop a model that accurately predicts RNA editing. In this study, we discovered multiple RNA sequence and secondary structure features important for A-to-I editing in Fusarium graminearum . We also identified the cis -sequence features with distinct importance for editing specificity and efficiency. The potential importance of full-length RNA molecules for editing efficiency is also revealed. This study represents the first comprehensive investigation of the cis -regulatory principles of A-to-I RNA editing in fungi. 

Summary Ascospores are the primary inoculum inFusarium graminearum. Interestingly, 70 of its genes have premature stop codons (PSC) and require A‐to‐I editing during sexual reproduction to encode full‐length proteins, including the ortholog of yeast Ama1, a meiosis‐specific activator of APC/C. In this study, we characterized the function ofFgAMA1and its PSC editing.FgAMA1was specifically expressed during sexual reproduction. TheFgama1mutant was normal in growth and perithecium formation but defective in ascospogenesis. Instead of forming four‐celled, uninucleate ascospores,Fgama1mutant produced oval, single‐celled, binucleated ascospores by selfing. Some mutant ascospores began to bud and underwent additional mitosis inside asci. Expression of the wild‐type or editedFgAMA1but not the uneditable allele complementedFgama1. In theFgama1xmat‐1‐1outcross, over 60% of the asci had eightFgama1or intermediate (elongated but single‐celled) ascospores, suggesting efficient meiotic silencing of unpairedFgAMA1. Deletion ofFgPAL1, one of the genes upregulated inFgama1also resulted in defects in ascospore morphology and budding. Overall, our results showed thatFgAMA1is dispensable for meiosis but important for ascospore formation and discharge. InF. graminearum, whereas some of its targets are functional during meiosis, FgAma1 may target other proteins that function after spore delimitation.