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.

subsequently mapped to the C. cinerea genome (NCBI assembly accession:

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Dicer-like proteins knockdown mediated by siRNAs of sRNA clean reads from both stages was 20-22 nt in length (Fig 1a) and had a 285 strong preference for 5' uracil (Fig 1b).   Table   302 2. The 20 and 26 nt classes were the most abundant groups in the milRNA candidates 303 (Fig 2a). Guanine dominated the 5' end nucleotide with weak superiority (Fig 2b).

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Similar to canonical miRNAs in animals and plants, most of the milRNAs in C.

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cinerea were derived from the intergenic region (68%), with five from rRNA (23%) 306 and two from exon (9%) (Fig 2c). As for the gene locations, half of the putative 307 milRNAs dwelled on the assembled chromosomes (Table 2). Six putative milRNAs 308 predicted in C. cinerea were located within a short distance on the U413 contig, 309 similar to milRNAs in animals and plants, which usually appear in clusters [60].

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Although no annotated genes were identified in the locus of mature cci-milR-12c,

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AGO-like-2 was expressed significantly higher in PRI than MYC. Therefore, DCL-1 406 or DCL-2 and AGO-like-2 are more likely involved in the biogenesis of cci-milR-12c.

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To fully understand the functions of the putative targets of milRNA, the targets were 479 annotated using GO terms, KOG terms and KEGG pathway. Results of GO term 480 annotation revealed that the majority (> 60%) of the putative targets of milRNAs were 481 categorized to the biological processes (Figs 10a and 10b). For both validated 482 milRNAs, "metabolism", "nucleobase, nucleoside, nucleotide and nucleic acid 483 metabolism", and "biosynthesis" were the most remarkably enriched GO terms under 484 this category (Fig 10c). Additional functional annotation of putative milRNA targets 485 was performed by searching the eukaryotic homologs in the KOG database. Putative (23%) was the largest group, followed by the "secondary metabolites biosynthesis, 490 transport and catabolism" (15%) category (Fig 11).

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Overall, results from the GO and KOG term annotations of the two validated 503 milRNAs were similar. None of the targets of cci-milR-12c was assigned to the 504 KEGG pathways and putative targets of cci-milR-13e-5p were annotated to 130 505 different pathways, most of them were classified into "metabolic pathways",