RNA-Seq, Bioinformatic Identification of Potential MicroRNA-like Small RNAs in the Edible Mushroom Agaricus bisporus and Experimental Approach for Their Validation

Abstract

Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.

Keywords: Agaricus bisporus; RT-qPCR; fungi; miRNAs; milRNAs; white button mushroom.

Figure 1

Length distribution of reads after mapping. (A) Total mapped reads. (B) Unique mapped reads.

Figure 2

Read distribution on chromosomes (Ch) and mitochondrion (Mt) after mapping. (A) Distribution of total reads before and after removing ncRNAs. (B) Distribution of unique reads before and after removing ncRNAs.

Figure 3

Small RNA loci annotation. Pie graphs show percentage of intergenic, exonic and intronic annotated reads. (A) Total reads. (B) Unique reads.

Figure 4

Secondary structures proposed for abi_milRNA precursors (abi_milRNA_1a, 2a, 4a and 6a are representative of those predicted by animal criteria and 8a and 17a of those with high homology with other fungi). The corresponding mature milRNA sequence and the estimated free energy of the pre-miRNAs are also indicated. Blue arrow: start mature sequence. Red arrow: end mature sequence.

Figure 5

Sequence logo of predicted A. bisporus milRNAs. Logo represents score of weighted counts from a multiple alignment.

Figure 6

Verification of pre−miRNAs and miRNAs by RT−qPCR. (A) Differential expression of abi_milRNA_1a, abi_milRNA_2a and abi_milRNA_4a pre−miRNAs in stipe (S) and cap (C). (B) Differential expression of abi_milRNA_1a, abi_milRNA_2a and abi_milRNA_4a mature miRNAs in stipe (S) and cap (C). *: Results are statistically significant at α: 0.1.

Figure 7

Flow-chart of the basic analysis process of milRNA sequencing data.