Abstract
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.
Highlights
IntroductionSmall non-coding RNAs (sRNAs), about 20–30 nucleotides (nt) in length, are the regulators of RNA interference (RNAi), a conserved eukaryotic gene silencing mechanism [1]. small RNA (sRNA) are categorized into three groups based on their origin and functions: small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs) and microRNAs (miRNAs) [2]
Small non-coding RNAs, about 20–30 nucleotides in length, are the regulators of RNA interference (RNAi), a conserved eukaryotic gene silencing mechanism [1]. small RNA (sRNA) are categorized into three groups based on their origin and functions: small interfering RNAs, piwi-interacting RNAs and microRNAs [2]
There were a limited number of putative targets of cci-milR-12c that were annotated with the functional Gene Ontology (GO) and KOG terms and were differentially expressed in MYC, some fruiting body related genes were included, including fungal pheromone, hydrophobin and cytochrome P450. These results suggest that miRNA-like RNAs (milRNAs) may play an important role on regulating different metabolic pathways and facilitating the cellular developments during the early developmental transition in C. cinerea
Summary
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 [1]. sRNAs are categorized into three groups based on their origin and functions: small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs) and microRNAs (miRNAs) [2]. 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 [1]. RNAi-related machineries are mainly responsible for genomic defence, heterochromatin formation and gene regulation [3]. MiRNAs are present in most eukaryotic lineages. They play essential roles in various biological processes by mediating post-transcriptional gene silencing to regulate gene expression through base pairing their seed region (2–7 nt at the 5’-end) to the untranslated region (UTR) or opening reading frame of their target genes [6,7,8,9,10,11,12,13]. MilRNAs have been subsequently discovered in other fungi, such as Sclerotinia sclerotiorum, Trichoderma reesei, Penicillium marneffei, Fusarium oxysporum, and Antrodia cinnamonmea, the potential roles of milRNAs in the developmental processes of mushroom forming fungi are still largely unknown [16,17,18,19,20]
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