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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. 

ABSTRACT In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in Aspergillus is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus Aspergillus nidulans . To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination. IMPORTANCE Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans . Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus .