Discovery of microRNA-like RNAs during early fruiting body development in the model mushroom Coprinopsis cinerea

Coprinopsis cinerea is a model mushroom particularly suited for the study of fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) have been extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), one for Argonaute (AGO), one for AGO-like and one for quelling deficient-2 (QDE-2) proteins, were also identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to those in animals and plants. Taken together, our findings provided the first evidence for milRNAs in the model mushroom and their potential roles in regulating fruiting body development. New information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights for guiding further functional and evolutionary studies of miRNAs.