Hindawi International Journal of Genomics Volume 2019, Article ID 9702342, 12 pages https://doi.org/10.1155/2019/9702342 Research Article Distribution, Characteristics, and Regulatory Potential of Long Noncoding RNAs in Brown-Rot Fungi Alessandra Borgognone ,1 Walter Sanseverino ,1 Riccardo Aiese Cigliano ,1 and Raúl Castanera 2 1 Sequentia Biotech, Edifici Eureka, Campus UAB, Cerdanyola del Vallès, Barcelona, Spain 2 Center for Research in Agricultural Genomics, CRAG (CSIC-IRTA-UAB-UB), Campus UAB, Cerdanyola del Vallès, Barcelona, Spain Correspondence should be addressed to Raúl Castanera; raul.castanera@cragenomica.es Received 13 January 2019; Accepted 7 March 2019; Published 2 May 2019 Academic Editor: Marco Gerdol Copyright © 2019 Alessandra Borgognone et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Long noncoding RNAs have been thoroughly studied in plants, animals, and yeasts, where they play important roles as regulators of transcription. Nevertheless, almost nothing is known about their presence and characteristics in filamentous fungi, especially in basidiomycetes. In the present study, we have carried out an exhaustive annotation and characterization of lncRNAs in two lignin degrader basidiomycetes, Coniophora puteana and Serpula lacrymans. We identified 2,712 putative lncRNAs in the former and 2,242 in the latter, mainly originating from intergenic locations of transposon-sparse genomic regions. The lncRNA length, GC content, expression levels, and stability of the secondary structure differ from coding transcripts but are similar in these two species and resemble that of other eukaryotes. Nevertheless, they lack sequence conservation. Also, we found that lncRNAs are transcriptionally regulated in the same proportion as genes when the fungus actively decomposes soil organic matter. Finally, up to 7% of the upstream gene regions of Coniophora puteana and Serpula lacrymans are transcribed and produce lncRNAs. The study of expression trends in these gene-lncRNA pairs uncovered groups with similar and opposite transcriptional profiles which may be the result of cis-transcriptional regulation. 1. Introduction alternative splicing, polyadenylation, and posttranscriptional modifications [4], but usually have little or no open reading In recent years, RNA sequencing (RNA-seq) has emerged as frames (ORF) [5]. They can be transcribed from either strand a powerful technology for genome-wide expression profiling. and are often classified based on their genome location and Advancements in high-throughput sequencing approaches context as sense, antisense, bidirectional, intronic, or inter- and reduction in costs led to the discovery of large amounts genic [6, 7]. An initial debate referred to these functional of RNA transcripts, including novel classes of non-protein- molecules as transcriptional “noise” [8]. However, in the coding transcripts. This revolutionary breakthrough helped last decades lncRNAs have gained importance as a heteroge- to unmask the noncoding fraction of eukaryotic transcrip- neous group of noncoding transcripts involved in diverse tomes, allowing the identification of long noncoding RNAs regulatory processes. As an example, recent experimental (lncRNAs) [1], a new functional class of RNAs pervasively findings indicated that Xist lncRNA is strictly required for transcribed in a wide range of genomes—from yeasts to X chromosome inactivation in animals [9], highlighting the animals. lncRNAs are generally defined as 200 nt or longer key role of lncRNAs in developmental processes such as transcripts, mostly transcribed by RNA polymerase II and embryogenesis and cell differentiation [10]. In addition, their characterized by very low or no coding potential [2, 3]. essential involvement in diverse cellular mechanisms associ- Similarly to the canonical structural processing of mRNA ated to transcriptional and posttranscriptional regulation, molecules, some lncRNAs are characterized by 5 ′ capping, protein translation, and chromatin modification [11, 12], 2 International Journal of Genomics among others, is becoming evident. While many research the repressive effects on transcription described for most efforts have been made to decipher the role of lncRNAs, less lncRNAs, HAX1 acts as an activator in the regulatory net- is known about their origin and precise molecular function. work of T. reesei cellulase expression. Also, the tight link A number of hypotheses attempted to elucidate the evolu- observed between a strain-specific variation of HAX1 length tionary pattern that led to the emergence of lncRNAs, sug- and its regulatory activity suggested a unique strategy for gesting their possible origin from alterations of protein- HAX1 dynamics [33]. To date, several studies have explored coding genes, modification of transposable elements, and the presence of lncRNAs in ascomycetes, but very little is activation of untranscribed DNA sequences [13, 14]. The fas- known about the distribution and properties of lncRNAs ter evolutionary turnover of lncRNAs compared to coding in basidiomycetes. A genome-wide characterization in the gene sequences and low degree of interspecies conservation white-rot Ganoderma lucidum identified a total of 402 across the eukaryotic domain [15] made difficult the identifi- putative lincRNAs (long intergenic noncoding RNAs), using cation of relationships between lncRNAs in organisms that RNA-seq data obtained from three developmental stages do not share a common evolutionary origin. Up to now, most combined with a modified 3 ′ RACE method to determine conservation studies were based on the analysis of primary the transcriptional direction of a transcript. These findings sequence. Nevertheless, more comprehensive approaches revealed the coexpression of a lincRNA subset with adja- attempted to integrate information from secondary struc- cent protein-coding genes, suggesting a potential lincRNA- tures and the functional role of lncRNAs to characterize the mediated transcriptional regulation of genes involved in evolutionary conservation on a global scale [15]. These strat- wood degradation, triterpenoid biosynthesis, and mating in egies are particularly relevant in light of the tight correlation G. lucidum [34]. between structure and function of most lncRNAs. This In this study, we carry out an in silico genome-wide emerged, for instance, by the discovery of their physical asso- characterization of lncRNAs in the basidiomycete fungi ciations (in cis or trans) with chromatin complexes required Coniophora puteana and Serpula lacrymans (also known for the epigenetic regulation of target regions [7, 16]. Consid- as “cellar” and “dry-rot” fungi, respectively) using transcrip- ering the increasing number of lncRNAs characterized across tomic data obtained from previous RNA-seq experiments. In eukaryotic genomes [17–19], important efforts have been their natural habitat, C. puteana and S. lacrymans produce made by researchers to describe and validate their functional brown-rot decay on the wood of conifers and occasion- role. While the discovery and characterization of lncRNAs in ally on hardwood trees. Additionally, both fungi are widely the animal and plant kingdoms is a current topic [18, 19], known because they cause damage on buildings and con- their abundance, features, and functional role remain poorly struction materials. In the last years, the genomes of a diverse investigated in fungi. Attempts to categorize lncRNAs in fun- group of Boletales species have been sequenced and anno- gal species have been based on different criteria, such as tated and shown to display important differences in terms mode of action in trans or cis, functional context, or addi- of genome size and gene content [35, 36]. Moreover, a recent tional properties [17]. In the last years, studies carried out study focused on the transposon landscape in Boletales in the budding yeast Saccharomyces cerevisiae and fission showed that TEs, especially LTR retrotransposons, have yeast Schizosaccharomyces pombe have elucidated the crucial undergone differential expansions leading to a great genomic regulatory role of lncRNAs in complex biological mecha- variability in this basidiomycete order [37]. nisms, which can act by interfering with the expression of sense or antisense proximal located genes. In budding yeasts, 2. Materials and Methods lncRNAs involved in the regulation of sexual sporulation can act by blocking aberrations during germ cell differentiation 2.1. Genome and Transcriptome Data Sources. S. lacrymans and interfering with sense transcription of genes involved and C. puteana raw mRNA-seq reads were obtained from in meiotic division [20, 21]. Although no evidence of such SRA database BioProject accession PRJNA272430. The data conserved regulatory mechanisms has been found in fission corresponds to mRNA obtained from mycelia cultured for yeast, lncRNAs also play crucial roles during the meiotic 7 days at 18°C in the dark in two different media using 3 development in these species, by blocking RNA elimination biological replicates: (i) MMN (Melin–Norkrans minimal factors that repress meiotic gene transcription [22, 23], facil- medium) and (ii) SOM (soil organic matter) extract (see itating proper homologous chromosome pairing [24], and [38] for details). This last medium consists of an MMN mediating the progression instead of the induction of meio- medium without nitrogen induced with a SOM extract sis [25]. Beside meiosis and mitosis, other regulatory strate- obtained from upper soil layer. gies of lncRNA are associated to a number of cellular processes in yeasts, such as cell-cell adhesion during filament 2.2. lncRNA Detection and Classification. FastQC was used formation of cells in response to nutrient starvation [26], for evaluating read quality and BBDuk tool from the BBMap regulation of phosphate metabolism in [27], stress response package (https://sourceforge.net/projects/bbmap/) for adapter [28], and telomere synthesis and maintenance [29, 30]. Fun- and quality trimming. A total of 77 clean million reads (50 bp) gal lncRNAs were also screened in other ascomycetes such obtained by merging replicates of the two conditions were as Neurospora crassa [31] and Fusarium graminearum [32], pooled and assembled with Trinity [39] (200 bp length cut- but the first functional characterization of a lncRNA in off), in order to obtain the reference transcriptomes of S. filamentous fungi (HAX1) was achieved only very recently lacrymans and C. puteana. The pipeline shown in Figure 1 in the saprophyte Trichoderma reesei [33]. Contrarily to was used for identifying lncRNAs. Briefly, TransDecoder International Journal of Genomics 3 Raw reads Mapping Trimming Subsampling Transcriptome assembly Differential expression 15 Trinotate proteins 10 BLASTx hits LogFC 5 0 CPC coding transcripts −5 −10 0 5 10 15 ncRNAs LogCounts Rfam ncRNAs lncRNAs Gene transcripts Figure 1: Pipeline of lncRNA annotation. [39] was run to identify candidate coding regions, which 3. Results and Discussion were filtered out. BLASTX searches [40] vs. each of the species reference annotation available in the Mycocosm 3.1. Genome-Wide Identification of lncRNAs in C. puteana database [41] were run with the remaining transcripts to and S. lacrymans. We assembled a total of 19,604 and identify known coding regions not detected by TransDecoder 14,103 isoforms in C. puteana and S. lacrymans, which were (i.e., small gene fragments). The remaining transcripts were later mapped to their respective genomes. We observed that scanned with CPC (coding potential calculator) [42], the assembled transcripts overlapped with 80.1 and 82% of retaining only those classified as noncoding. This dataset the genes present in the reference annotations of C. puteana was then used as query against Rfam database using Infer- and S. lacrymans [35, 50]. In addition, 77.8% and 86.9% of nal software [43] to remove known noncoding RNAs other these overlapping transcripts were potentially full-length than lncRNAs. The remaining noncoding transcripts were (according to a reciprocal overlap between reference gene mapped to the genome with gmap [44] with a 90% identity and assembled isoform higher than 80% of their respective cut-off, and only the best hit was reported. lengths). Considering that some annotated genes have no transcript support (i.e., gene models built based only on homology or structural evidence) and some others might 2.3. Characterization of Minimum Free Energy. Minimum not be expressed in the tested conditions, this evidence sug- free energy (MFE) values for lncRNAs and protein-coding gests that our assemblies captured a very important fraction RNAs were estimated using the RNAfold algorithm from of the transcriptomes. Nevertheless, the assembly of S. lacry- the ViennaRNA package [45]. The program was run using mans was slightly more comprehensive than that of C. the following parameters: -p –d2 –noLP. MFE of 1,000 ran- puteana. In addition, we found that only 5.3 and 4.5% of domly selected lncRNAs and protein-coding transcripts were the assembled coding transcripts (Figure 1, “gene tran- compared after normalizing by transcript length. scripts”) did not overlap with genes in C. puteana and S. lacrymans, respectively. These transcripts may represent 2.4. Transcriptome Quantification. Libraries were mapped gene fragments or actual genes specifically expressed in the back to the corresponding transcriptome assembly using conditions assayed. Also, the low number of transcripts in Bowtie2 [46], and expression levels of annotated features this category evidences that the official annotations are very were calculated using RSEM [47] in TMM-normalized exhaustive. After discarding coding transcripts as well as TPM values (transcripts per million). Differential expres- other types of noncoding RNAs using our pipeline, we iden- sion of lncRNAs between the two conditions was obtained tified 2,712 lncRNAs in C. puteana and 2,242 in S. lacrymans, with EdgeR [48] using a false discovery rate < 0 05 and a (Supplementary Datasets S1 and S2). The vast majority of LogFC > 1. lncRNAs originated from intergenic regions in both species (76 and 85% in C. puteana and S. lacrymans, respectively), whereas 14 and 22% overlapped with exons, and less than 2.5. Interspecies Conservation of lncRNA. All-by-all BLASTN 2% were intronic (Table 1). A previous genome-wide study followed by MCL (Markov cluster) clustering [49] (inflation focused on lncRNAs in the basidiomycete G. lucidum value = 2) was performed with S. lacrymans and C. puteana reported 402 long intergenic ncRNAs [34]. Also, a study putative lncRNAs. Conserved lncRNAs were identified by carried out in Cryptococcus neoformans identified 1,197 tran- looking for clusters with members of both species. scribed regions without coding potential ranging from 106 to 4 International Journal of Genomics Table 1: Classification of assembled transcripts in C. puteana and S. monoexonic (60-65%) whereas 20-25% displayed 2 exons lacrymans. (Figure 3). In fact, as exon length was similar in lncRNAs and genes of both species (ranging from 239 to 275 nt in Feature C. puteana S. lacrymans C. puteana and 250 to 261 in S. lacrymans), the differences Isoform 19,604 14,103 in transcript length between the lncRNAs and genes could Isoform clusters∗ 17,208 12,543 be mainly due to the higher exon number of coding elements. Coding isoform 16,279 11,345 Similar features were described in the ascomycete F. grami- lncRNA (all) 2,712 2,242 nearum during perithecial development, which displayed a much higher number of monoexonic lncRNAs than lncRNA intergenic 2,059 1,912 mRNAs (~70% vs. 20%) and transcript lengths ranging from lncRNA overlapping exon 607 314 200 to 2,000 nt approximately. In addition, the AU content of lncRNA intronic 46 16 lncRNAs in this filamentous fungus was higher than coding ∗ sequences of mRNAs, but slightly lower than intergenic Cluster of isoforms originating from a single locus. regions [32]. Previous studies in humans and plants have shown that 5,555 nt [51]. In ascomycetes, recent studies identified a lncRNAs are substantially less folded than mRNAs [57, 58]. similar content of lncRNA candidates, as described for F. The stability of a secondary structure and backbone confor- graminearum (547 developmental stage-specific lncRNAs) mation of an RNA molecule is determined by its minimum [32] and N. crassa (939 and 1478 lincRNAs [31, 52]). In S. free energy (MFE), with lower energy values being associ- cerevisiae, approximately 2,000 lncRNAs have been identi- ated to more stable structures [59]. To estimate the MFE of fied thus far and their presence and abundance have been putative lncRNAs and compare it with that of coding tran- linked to different growing conditions [53, 54]. In this sense, scripts, we applied the RNAfold algorithm [45] to 1,000 ran- it should be noted that the gene content and genome size domly selected transcripts from each group (lncRNAs and of the latter species are much lower than C. puteana or S. coding), and the results were corrected by the sequence lacrymans, and thus the density of lncRNAs is clearly length. We found that lncRNAs display higher corrected higher in spite of the similar number of lncRNAs detected. MFE than do genes in both species (Figure 4, Wilcoxon Our results are in line with the very few findings dealing p value = 2.2e-16), indicative of a lower thermodynamic sta- with basidiomycetes lncRNAs and suggest that these tran- bility. As reported in previous studies, a secondary structure scriptional units represent a much smaller fraction of the of lncRNAs tends to have less stable conformation and con- fungal transcriptome than the coding transcripts. This con- sequently higher free energy than do protein-coding tran- trasts to what has been described for humans, where up to scripts [57]. Similar findings were observed in the plant 68% of the genes are noncoding [55] and might be explained pathogen oomycete Phytophthora sojae, where 940 identified by the shorter intergenic distances of fungal genomes in lncRNAs displayed higher MFE mean values when com- comparison to other eukaryotes, which are the main source pared to protein-coding genes (-191 vs. -214 kcal/mol) [60]. of lncRNAs. In spite of the similar characteristics found in S. lacrymans and C. puteana lncRNAs in terms of quantity, length, exon 3.2. C. puteana and S. lacrymans Elements Share the number, or secondary structure stability, we found almost Signatures of Canonical Eukaryotic lncRNAs. The vast major- no sequence conservation among them. After clustering by ity of lncRNAs were shorter than 500 nt in both species similarity all the sequences using all-by-all BLASTN followed (Figure 2(a)), with S. lacrymans lncRNAs being longer than by MCL, we found only three small clusters displaying those of C. puteana (mean of 434 nt vs. 377 nt, Wilcoxon p lncRNAs from the two species (one with three sequences value = 1.288e-08). The same pattern was found on coding and the remaining with only two). The low sequence conser- sequences (1,889 nt vs. 1,612 nt), although we cannot rule vation of lncRNAs has been long known in plants and ani- out the possibility that these small but significant differences mals, although this phenomenon has not been investigated are due to the assembly process. Eukaryotic lncRNAs are far in fungi due to the limited amount of available information. shorter than genes and by definition are longer than 200 nt, A hypothesis to explain such low conservation levels is although noncoding transcripts much longer that this that the selection may act on structure rather than on pri- threshold are sometimes found. In example, a study analyz- mary sequence [61]. Nevertheless, further experimental ing lncRNAs in the basidiomycete Ganoderma lucidum work will be needed to demonstrate whether this phenom- reported an average length of 609 nt and an important frac- enon occurs in filamentous fungi. It has been proposed tion of lncRNAs longer than 1,000 bp [34]. In terms of GC that the low similarity of lncRNAs identified in F. grami- content, lncRNAs of both species showed a lower content nearum with those found in other eukaryotes might reflect than genes (Figure 2(b)). This phenomenon has been linked the nonexhaustive annotation status of lncRNAs in fila- to a higher accessibility for putative interactions with cellular mentous fungi or the high sequence divergence in fungal factors [56]. S. lacrymans lncRNAs displayed the lowest CG lncRNA [32]. In this sense, our results point to a general content, with an average GC value per element of 48.5% vs. lack of sequence conservation of lncRNAs in the fungal the 54.9% found for C. puteana. This finding does not only kingdom. The estimated divergence between the two species apply to lncRNAs, as coding sequences follow the same was dated at 84 million years ago [36], but as brown-rot trend. In addition, lncRNAs from both species were mainly fungal species they share a lifestyle and have a conserved International Journal of Genomics 5 C. puteana S. lacrymans 4000 2000 0 Count 4000 2000 0 0 5000 10000 15000 0 5000 10000 15000 Length Gene lncRNA (a) 70 60 GC content 50 40 30 C. puteana genes C. puteana lncRNAs S. lacrymans genes S. lacrymans lncRNAs Group Genes lncRNA (b) Figure 2: Distribution of length (a) and percentage of GC content (b) of coding isoforms and lncRNAs in C. puteana and S. lacrymans. 6 International Journal of Genomics 0 20 40 60 for plants or animals, where 20-80% of the lncRNAs have a Gene lncRNA TE origin [13, 64, 65]. This fact is even more striking in the case of S. lacrymans, as this species has undergone a dramatic amplification of RNA transposons during the last 5 million 7 years, especially of LTR retrotransposons in the Gypsy super- family [37]. LTR retrotransposons are the most prevalent TE order in fungi [66], and also in most plants [67]. Their ability to efficiently colonize host genomes reside in their copy-and- 6 paste mechanism (in contrast to the cut-and-paste mecha- nism of most DNA transposons) but also in the fact that they tend to accumulate in gene-poor heterochromatic 5 regions. Most of the LTR retrotransposons of S. lacrymans are clustered in transcriptionally silent regions displaying very few lncRNAs (Supplementary Figure S3). Previous studies in other basidiomycetes such as Coprinopsis cinerea, Exon 4 Laccaria bicolor, or Pleurotus ostreatus have demonstrated that transposon-rich regions are specifically targeted by DNA methylation and siRNAs, which efficiently shut down 3 their expression [68, 69]. In this sense, the lack of lncRNAs in the repeat-rich regions of the genome seems to be a side effect of the arms race between TEs and fungal genome defense mechanisms. 2 3.4. Transcriptional Profiles of lncRNA. The expression levels of lncRNAs were lower than those of coding transcripts 1 (Figure 5). As an example, in SOM medium, S. lacrymans lncRNAs had an average expression of 12.8 TPM vs. 89.2 TPM of coding transcripts. In the same conditions, C. puteana lncRNAs displayed an average of 15.6 TPM vs. 0 20 40 60 58.9 TPM of coding transcripts. This phenomenon has been Percentage previously described for many plant and animal lncRNAs, C. puteana and it is in fact considered a canonical feature of these S. lacrymans pervasive transcripts [2]. The global distribution of transcrip- tion levels was very similar in C. puteana and S. lacrymans Figure 3: Exon number of lncRNAs and coding isoforms in C. puteana (orange) and S. lacrymans (yellow). and independent of the culture media used (Figure 5, Supple- mentary Figure S4). In addition, the expression of lncRNAs was found to be more uniform than that of genes, with the lignin-degrading machinery based on common extracellular levels of most transcripts concentrated around the median enzymes [62, 63]. Nevertheless, the important differences of the population (Figure 5). In addition to the general found in the dynamics of transposable elements [37] and expression levels, we performed differential expression the absence of lncRNA conservation suggest that the evolu- analyses to identify lncRNAs showing a significant increase tion of the noncoding genome is highly divergent in these or decrease in expression when the fungus grows on media two species. with organic matter extracts (SOM medium). Using a log2- fold change cutoff of 1 and an FDR adjusted p value <0.05, 3.3. Transposon-Associated lncRNAs. The coordinates of we identified 341 (12.6%) differentially expressed lncRNAs annotated lncRNAs were intersected with REPET TE anno- between the two conditions in C. puteana and 210 (9.4%) tations of both species [37] to uncover the presence of TE- in S. lacrymans. A total of 184 lncRNAs were upregulated associated lncRNAs. In the case of C. puteana, only 62 out in SOM medium and 157 downregulated in the former, of the 2,712 annotated lncRNAs overlapped with an anno- whereas the latter showed 89 SOM-upregulated lncRNAs tated TE. A total of 40 TE families had at least one lncRNA and 121 downregulated. The proportions of DE-lncRNAs overlapping with any of their members. Remarkably, 75% are similar or even higher than those found for coding of these TE families producing lncRNAs were DNA ele- transcripts using the same cutoff (9.9% in C. puteana and ments, especially TIR (terminal inverted repeat) elements 10.0% in S. lacrymans). The presence of differential tran- and MITEs (miniature inverted-repeat transposable ele- scription suggests that these elements are likely functional ments). In S. lacrymans, we found 64 out of the 2,242 and not simply the result of transcriptional noise. Differ- lncRNAs overlapping TEs (38 TE families in total), with the entially expressed lncRNAs have been widely studied in same prevalence of DNA transposons over RNA transposons humans, where a fraction of them show transcriptional (63% of the TE families of the DNA class). This amount of profiles associated to different cancer types [55]. In plants, TE-associated lncRNAs is much lower than that reported many lncRNAs show stage-specific transcriptional profiles, International Journal of Genomics 7 C. puteana S. lacrymans 15 10 Density 5 0 −0.5 −0.4 −0.3 −0.2 −0.1 0.0 −0.5 −0.4 −0.3 −0.2 −0.1 0.0 kcal/mol/nt Genes lncRNA Figure 4: Minimum free energy (kcal/mol/nt) of 1,000 random lncRNAs and coding transcripts normalized by length. 15 10 Log2 (TPM + 0.001) 5 0 −5 −10 C. puteana - genes C. puteana - lncRNAs S. lacrymans - genes S. lacrymans lncRNAs Class C. puteana - genes S. lacrymans - genes C. puteana - lncRNAs S. lacrymans - lncRNAs Figure 5: Distribution of transcriptional levels of coding transcripts and lncRNAs of C. puteana and S. lacrymans cultured on SOM medium. Box plots inside the violin plots are presented, showing the median value as a horizontal line. suggesting a putative role during reproductive development activation and repression of genes involved in the [70]. In fungi, a study reported the presence of 11 lncRNAs metabolism of sugars other than glucose [71]. Also, Shah upregulated in response to light, whereas no differential et al. report that 16 to 29% of the genes of a set of 10 expression was found in response to temperature [52]. It is brown-rot and ectomycorrhizal fungi were upregulated in well-known that fungi adjust their transcriptome in response SOM media [38]. According to our results, the noncoding to nutrient stimuli. In example, S. cerevisiae undergoes mas- transcriptome is also regulated in response to nutrient sive transcriptional reprogramming as a consequence of availability. We spec-ulate that the presence of differentially 8 Log2 FC (SOM/MNN) Log2 FC (SOM/MNN) −3 0 3 6 −4 −2 0 2 protID_84488 protID_140112 protID_145622 Type protID_159631 Type protID_99654 protID_123937 protID_100918 Gene protID_91786 Gene protID_157963 protID_119590 lncRNA lncRNA protID_82255 protID_164374 protID_137501 protID_126084 protID_81112 protID_88282 protID_166335 protID_76224 protID_112255 protID_139557 protID_70356 protID_51642 protID_141671 protID_120194 protID_84584 protID_114063 protID_157164 protID_113207 protID_156620 protID_96686 protID_168431 protID_135329 protID_148000 protID_137243 protID_140570 trends in SOM vs. minimal MNN media. protID_134730 protID_102654 protID_156948 protID_166004 protID_139415 protID_109602 protID_85682 protID_123715 protID_167256 protID_120915 protID_145080 protID_139802 protID_65796 protID_144997 protID_139755 protID_51805 protID_85739 protID_137237 protID_170071 protID_135351 protID_148123 protID_109263 protID_127584 protID_44842 protID_80341 protID_79566 protID_124807 protID_50502 protID_72825 protID_137434 fungus is under active decomposition of soil organic matter. genes and 902 S. lacrymans genes of their corresponding 3.5. lncRNAs in Brown-Rot Fungi: A Source of cis-Regulatory S. lacrymans [50]. We found that up to 1,011 C. puteana expressed lncRNAs in C. puteana and S. lacrymans in SOM respect of the reference annotations of C. puteana [35] and of our annotated lncRNAs, we analyzed their position in media may underlie a function of these elements when the Elements? In order to investigate the cis-regulatory potential protID_140640 protID_51049 protID_67148 protID_156542 protID_68594 (a) (b) protID_74514 protID_141444 protID_71749 protID_83675 protID_77306 protID_167088 protID_126365 protID_76230 protID_73002 protID_98697 protID_17575 protID_170062 protID_163923 protID_157281 protID_125023 protID_147024 protID_76265 protID_112511 protID_119024 protID_80384 protID_59509 protID_58544 protID_53947 protID_130409 protID_128915 protID_138853 protID_164433 protID_156504 protID_126359 protID_165636 protID_129313 protID_144499 protID_112319 protID_111433 protID_143972 protID_123858 protID_58783 protID_139667 protID_37063 protID_91912 protID_131925 protID_52592 protID_167159 protID_136346 protID_165406 protID_166414 protID_147842 protID_164316 protID_50909 protID_85883 protID_162055 protID_64346 protID_86811 protID_148028 protID_131240 protID_133649 protID_104209 protID_145980 protID_148034 protID_134067 protID_98130 protID_168507 protID_76620 protID_101154 protID_120606 protID_91504 protID_84437 protID_165887 protID_90031 protID_21818 protID_166338 protID_76744 protID_57073 protID_152779 protID_76360 protID_54011 protID_64217 upstream lncRNA, we used the differentially expressed species and compared the gene expression trends (log2FC) puteana and S. lacrymans reference annotations) in both “lncRNA associated genes,” Supplementary Table S6). To Figure 6: Expression profiles of 147 C. puteana DE genes and their linked upstream lncRNAs, showing opposite (a) and same (b) expression International Journal of Genomics genes between SOM and MNN media published by Shah reference annotations have a lncRNA at less than 500 bp with those of our lncRNAs. The aim was to analyze if the test if these genes could be under the regulation of the upstream the transcription start site (hereafter referred as et al. [38] (original transcriptome datasets analyzed with C. International Journal of Genomics 9 Table 2: Top ten enriched terms in C. puteana and S. lacrymans DEG genes with lncRNAs in their promoter region. Ratio in Ratio in Species GO Category GO name DEG p value p value fdr genome genes GO:0006508 BP Proteolysis 8,8 2,1 1, 19E − 05 0,006 GO:0005975 BP Carbohydrate metabolic process 6,1 1,4 0,000202 0,076 GO:0006810 BP Transport 8,8 2,8 0,000224 0,076 GO:0000272 BP Polysaccharide catabolic process 1,4 0,0 0,00165 0,3 GO:0004190 MF Aspartic-type endopeptidase activity 4,8 0,7 5, 65E − 05 0,018 C puteana Hydrolase activity, hydrolyzing O-glycosyl GO:0004553 MF 4,8 0,9 0,000315 0,09 compounds GO:0005215 MF Transporter activity 6,1 1,7 0,000865 0,202 GO:0008810 MF Cellulase activity 1,4 0,0 0,00111 0,228 GO:0004338 MF Glucan exo-1,3-beta-glucosidase activity 1,4 0,1 0,00304 0,414 GO:0030246 MF Carbohydrate binding 2,0 0,2 0,00472 0,54 GO:0005975 BP Carbohydrate metabolic process 5,8 1,1 0,000289 0,088 GO:0006725 BP Cellular aromatic compound metabolic process 2,5 0,2 0,00272 0,41 GO:0016998 BP Cell wall macromolecule catabolic process 1,7 0,1 0,00374 0,47 GO:0008152 BP Metabolic process 11,7 5,3 0,0059 0,554 Hydrolase activity, hydrolyzing O-glycosyl 6, 87E − 06 S. GO:0004553 MF 5,8 0,6 0,01 compounds lacrymans GO:0016491 MF Oxidoreductase activity 10,0 3,1 0,000372 0,092 GO:0019131 MF Obsolete tripeptidyl-peptidase I activity 1,7 0,1 0,00236 0,378 GO:0003824 MF Catalytic activity 11,7 5,3 0,00561 0,548 GO:0018658 MF Salicylate 1-monooxygenase activity 1,7 0,1 0,00846 0,656 GO:0048037 MF Cofactor binding 2,5 0,4 0,00858 0,688 ∗ Ratios correspond to percentage of genes with this associated GO term. transcriptional response of the genes to the culture media was characterization of a lncRNA was recently published in the the same or the contrary than its upstream lncRNA. In C. human pathogen Cryptococcus, where RZE1 was demon- puteana, we found that 147 out of the 1,011 lncRNA- strated to control yeast-to-hypha transition, through the associated genes were differentially expressed (log2FC transcriptional cis-regulation in of the downstream gene cutoff = 1, FDR adjusted p value <0.05). The analysis of the ZNF2 [73]. Our results indicate that the upstream regions corresponding 147 gene-lncRNA pairs revealed 48 of them of about 7% of C. puteana and S. lacrymans genes are having an opposite expression and 99 of them showing transcribed in the form of lncRNAs, and some of these the same expression profile (Figure 6). In the case of S. genes might be positively or negatively regulated by them. lacrymans, 120 out of the 902 lncRNA-associated genes In a genome-wide study carried out in the white rot basid- were differentially expressed. After a closer analysis of the iomycete G. lucidum, the authors also found positive and 120 gene-lncRNA pairs, we found 45 showing an opposite negative correlations between lncRNAs and surrounding expression and 73 with the same expression trend (Supple- genes, some of them coding for enzymes involved in wood mentary Figure S5). lncRNAs are known cis-regulatory degradation [34]. When we analyzed the function of DE elements in plants and animals. The most recent literature genes under potential lncRNA regulation, we found that suggests that the expression of lncRNA is usually highly their associated GO terms were related to proteolysis and correlated (either positively or negatively) with that of their carbohydrate metabolism, which are typically enriched in surrounding genes. Positive correlations might result from the secretome of lignin-degrading species [37]. Never- common regulations of local chromatin or actual lncRNA theless, the enrichment of most of these GO terms was not regulation [70]. By contrast, the overexpression of a lncRNA significant after correction for multiple testing (Table 2). In may also lead to a downregulation in the expression of agreement with this finding, oxidoreductases, hydrolases, and adjacent genes by physical interference with transcription peptidases were described as the most upregulated secreted or by overlap with the proximal gene [17]. In yeasts, the enzyme genes during brown-rot and ectomycorrhizal fungi expression of the HO gene has been experimentally dem- growth on SOM media [38]. In this sense, future experi- onstrated to be regulated by the expression of a lncRNA in mental work will be needed to understand the link between the promoter region by displacement of a cell-cycle box transcription of basidiomycete lncRNAs and their linked binding factor [72]. In basidiomycetes, the first functional genes, especially those involved in wood degradation. 10 International Journal of Genomics 4. Conclusions showing opposite (A) and same (B) expression trends in SOM vs. minimal MNN media. In the present study, we have uncovered the presence of 2,712 and 2,242 putative lncRNAs in the basidiomycetes C. Supplementary 6. Table S6: annotation and expression ratios puteana and S. lacrymans, mainly arising from intergenic of gene-lncRNA pairs in C. puteana and S. lacrymans. regions. 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