Date Published: September 19, 2018
Publisher: Public Library of Science
Author(s): Amy Yuet Ting Lau, Xuanjin Cheng, Chi Keung Cheng, Wenyan Nong, Man Kit Cheung, Raymond Hon-Fu Chan, Jerome Ho Lam Hui, Hoi Shan Kwan, Erika Kothe.
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.
Small non-coding RNAs (sRNAs), about 20–30 nucleotides (nt) in length, are the regulators of RNA interference (RNAi), a conserved eukaryotic gene silencing mechanism . sRNAs are categorized into three groups based on their origin and functions: small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs) and microRNAs (miRNAs) . In fungi, RNAi-related machineries are mainly responsible for genomic defence, heterochromatin formation and gene regulation . For example, siRNAs mediate quelling and meiotic silencing of unpaired DNA as genomic surveillance against viral infection in Cryphpnectria parasitica, and against transposon invasion and silencing unpaired DNA in Neurospora crassa [3, 4, 5]. Most of the descriptions of RNAi pathways of sRNAs in various fungi have focused only on the siRNA-directed pathways.
In this study, we constructed sRNA libraries and identified milRNAs of C. cinerea at two different developmental stages. Characteristics of C. cinerea milRNA populations similar to those in animals and plants and the presence of core proteins of miRNA biogenesis in C. cinerea suggest that milRNAs in mushrooms may be produced in similar pathways to those in animals and plants. The functional analysis of milRNA targets also demonstrates the potential regulatory roles of milRNAs in fruiting body development (S2 Fig).
Our findings have demonstrated differential post-transcriptional regulatory roles of milRNAs in different developmental stages of the mushroom forming fungus C. cinerea and identified the milRNA potential targets involved in fruiting body formation, providing new insights into the regulatory mechanisms of fruiting body development and the potential functions of milRNAs in fungi. Moreover, we have found putative core miRNA biogenesis proteins, Dicer and AGO, in the C. cinerea genome. Phylogenetic analysis showed that these proteins were more closely related to those in other fungal species than to those in animals and plants. However, the roles of DCLs, AGO and QDE-2 proteins in the biogenesis of C. cinerea milRNAs cannot be shown here. Altogether, these results serve as the foundation for further evolutionary developmental studies of fungi and contribute to the phylogenetic occurrence of miRNA-mediated regulatory system among different kingdoms.