Abundant and selective RNA-editing events in the medicinal mushroom Ganoderma lucidum

Abstract

RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about genome-scale RNA editing in fungi. In this study, we screened for fungal RNA editing sites at the genomic level in Ganoderma lucidum, a valuable medicinal fungus. On the basis of our pipeline that predicted the editing sites from genomic and transcriptomic data, a total of 8906 possible RNA-editing sites were identified within the G. lucidum genome, including the exon and intron sequences and the 5'-/3'-untranslated regions of 2991 genes and the intergenic regions. The major editing types included C-to-U, A-to-G, G-to-A, and U-to-C conversions. Four putative RNA-editing enzymes were identified, including three adenosine deaminases acting on transfer RNA and a deoxycytidylate deaminase. The genes containing RNA-editing sites were functionally classified by the Kyoto Encyclopedia of Genes and Genomes enrichment and gene ontology analysis. The key functional groupings enriched for RNA-editing sites included laccase genes involved in lignin degradation, key enzymes involved in triterpenoid biosynthesis, and transcription factors. A total of 97 putative editing sites were randomly selected and validated by using PCR and Sanger sequencing. We presented an accurate and large-scale identification of RNA-editing events in G. lucidum, providing global and quantitative cataloging of RNA editing in the fungal genome. This study will shed light on the role of transcriptional plasticity in the growth and development of G. lucidum, as well as its adaptation to the environment and the regulation of valuable secondary metabolite pathways.

Keywords: Ganoderma lucidum; RNA editing; RNA-Seq.

Figure 1

Pipeline flowchart for calling RNA-editing sites. Raw inputs include RNA-Seq reads sequenced by 454 and Illumina and genomic 454-reads produced by diploid genome resequencing. These sequences were filtered and analyzed further in this pipeline.

Figure 2

Distribution of RNA-editing loci in fruiting bodies.

Figure 3

RNA-editing types and RNA-editing degree in different genomic regions. (A–D) Distribution of RNA-editing types on the gene models, including CDS (A), 5′-UTR (B), 3′-UTR (C), and introns (D). (E–F) Distribution of RNA-editing levels on the entire genome (E) and protein-coding regions (F).

Figure 4

Influence of RNA editing on amino acid substitutions in the coding regions. (A) Frequency of codon change at three codon positions. (B) Proportion of nonsynonymous and synonymous substitution of amino acids for different editing types. RNA-editing types above the horizontal dashed line indicate that the proportion of nonsynonymous substitution and synonymous substitution is >1.

Figure 5

Phylogenetic and domain analysis of ADATs and ADARs. (A) Unrooted tree of ADATs and ADARs. The number of bootstrap replications was set as 1000 for testing the confidence level of the phylogenetic tree. Only bootstrap values >50 are shown. (B) A schematic of ADAR and ADAT enzymes. The domain structures were highlighted by using different colors. dsRBD: double-stranded RNA-binding domain (PF00035); A_deamin: adenosine deaminase (PF02137); z-alpha: adenosine deaminase z-alpha domain (PF02295); dCMP_cyt_deam_1: cytidine and deoxycytidylate deaminase zinc-binding region (PF00383).

Figure 6

Validation of predicted RNA-editing sites by Sanger sequencing. (A-D) The chromatogram traces of both strands, which were generated from gDNA and cDNA, are shown. The blue vertical line indicates editing sites on both strands.

Figure 7

Comparison of RNA and protein secondary structures for RNA-editing transcripts. (A) RNA secondary structure of phosphoglucomutase involved in polysaccharide biosynthesis. RNA-editing sites were located at 306,124 and 306,136 of GaLu96scf_34. Minimum free energy was shown as the value of “dG.” Blue letters and arrows represent original bases or amino acids. Red letters and arrows represent edited bases or amino acids. (B) Protein secondary structure of phosphoglucomutase. For each protein, the α-helix (Alpha helix) represents protein secondary structures; the question mark represents disordered/unstructured amino acids; “confidence” refers to the confidence of secondary structure and disordered/unstructured amino acids. (C) The change of RNA secondary structure of mevalonate kinase in the mevalonate pathway. RNA-editing sites were located at 1,010,080 of GaLu96scf_8.