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Alternative splice type distribution per taxon from random sampling approach. Pie portions: intron retention (IR), skipped exons (SE), alternative 5′ splice sites (A5′SS) and 3′ splice sites (A3′SS). Only the 23 informative fungi are considered, i.e. those where AS rates could be estimated (cf. Table 1). Only non-yeasts are considered in chart 1B (17 ascomycetes − 3 yeasts = 14).
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Alternative splicing (AS) is a cellular process that increases a cell's coding capacity from a limited set of genes. Although AS is common in higher plants and animals, its prevalence in other eukaryotes is mostly unknown. In fungi the involvement of AS in gene expression and its effect on multi-cellularity and virulence is of great medical and eco...
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Context 1
... numbers of detected AS events strongly depend on the numbers of available ESTs (Supplementary Fig. S1a; Pearson correlation coefficient r ¼ 0.82, P-value 1.8 Â 10 26 ). A very high coverage of introns with ESTs, especially when using next generation transcrip- tome sequencing, can reveal even very rare events that may partly represent splicing noise of the cell. ...Context 2
... mapped the AS events that were recovered by random sampling to genomic locations of annotated genes to calculate AS rates per gene. We found that the correlation between these AS rates and the EST numbers is clearly reduced (r ¼ 0.16, P-value ¼ 0.46, Supplementary Fig. S1b). Thus, random sampling gives AS rate estimates that are com- parable between species. ...Context 3
... genes), the rate is 7.3%. Coccidioides immitis and C. neoformans show outstanding AS rates of 13 and 18/20%, respect- ively (strains JEC21 and B-3501A). The relative propor- tions of the AS types averaged over all species, in the order of frequency are: intron retention 61%, alterna- tive 3 0 SSs 23%, alternative 5 0 SSs 13% and skipped exons 3% (Fig. 1a). We only took into account strains B-3501A and Pb01 from C. neoformans and P. brasilien- sis, respectively, for mean value ...Context 4
... not sig- nificant, n ¼ 5/14). Without the species showing outly- ing AS rates (C. immitis, P. brasiliensis, C. neoformans), the rates for Basidiomycota (6.1%) and Ascomycota (4.9%) are still different. Basidio-mycota and Ascomycota have very similar AS type proportions, with Basidiomycota showing slightly more RIs and less alternative 5 0 SSs (Fig. 1). In both cases RIs make up around two-thirds of all AS events while skipped exons are only marginally present. The ascomycetous yeasts of our study (P. stipi- tis, S. cerevisiae and S. pombe) show an AS rate of 0.26% on average, which is significantly lower than the rate of the other Ascomycota (Mann -Whitney U- test, P-value 0.003, n ...Context 5
... in a recent study on fission yeasts, 433 AS events in overall 5144 genes were found in S. pombe. 35 While considering scaling effects due to sequencing depth, our results agree well with these findings in that the AS rate is very low compared with that in non-yeast Ascomycota (see Supplementary Calculation S1). This validates the comparability of our normalized AS rate results. ...Similar publications
Globally, foliar pathogenic fungi cause serious losses of annual and perennial crops, ornamentals, landscape plants and forest trees. Plant pathogens that infect foliage are a diverse assemblage of fungi representing both phyla: Ascomycota and Basidiomycota. Although most of the species found on living leaves have been well studied by mycologists a...
Citations
... Previous studies, including intron lariat sequencing, short-read RNA sequencing (RNAseq), and Iso-Seq, have unveiled many low-frequency alternative isoforms, indicating relatively low splicing fidelity in fission yeast [21][22][23]. However, regulated alternative splicing has not been observed as a major issue in mitotically active fission yeast cells [24]. Interestingly, certain genes seem to be regulated by splicing during sexual differentiation. ...
Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5’ splicing site (5’SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5’SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5’SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.
... Intron retention is the most commonly described AS event in fungi [14][15][16] . Even though the AS phenomenon has been known for over 40 years, the annotated transcripts only partially reflect the huge complexity of all AS events in fungi. ...
... Missing or incorrect genome annotations and the avail-ability of datasets only about the most popular gene transcripts in the fungal databases limit our current knowledge of the biological impact of AS 17 . Increasing the analysis of RNA-seq data suggests a critical role for AS in the fungal kingdom [14][15][16] . The histidine-containing phosphotransfer protein Ypd1p plays an important role in osmoregulation as part of the high osmolarity glycerol (HOG) pathway. ...
Magnaporthe oryzae is placed first on a list of the world’s top ten plant pathogens with the highest scientific and economic importance. The locus MGG_07173 occurs only once in the genome of M. oryzae and encodes the phosphotransfer protein MoYpd1p, which plays an important role in the high osmolarity glycerol (HOG) signaling pathway for osmoregulation. Originating from this locus, at least three MoYPD1 isoforms are produced in a signal-specific manner. The transcript levels of these MoYPD1-isoforms were individually affected by external stress. Salt (KCI) stress raised MoYPD1_T0 abundance, whereas osmotic stress by sorbitol elevates MoYPD1_T1 levels. In line with this, signal-specific nuclear translocation of green fluorescent protein-fused MoYpd1p isoforms in response to stress was observed. Mutant strains that produce only one of the MoYpd1p isoforms are less virulent, suggesting a combination thereof is required to invade the host successfully. In summary, we demonstrate signal-specific production of MoYpd1p isoforms that individually increase signal diversity and orchestrate virulence in M. oryzae.
... Post-transcriptional regulation through alternative splicing (AS) allows the production of various protein isoforms in response to physiological requirements and environmental cues, often serving as drivers of phenotypic diversity within the eukaryotic cell proteome [27][28][29]. Intron retention (IR) is one of the most common AS events in fungi [30,31], and it may be relevant in the regulatory mechanisms of fungal physiology, adaptation to fungal niches, pathogenicity, and drug resistance [13,32,33]. ...
Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum–host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.
... Previous studies, including intron lariat sequencing, short-read RNA sequencing (RNAseq), and Iso-Seq, have unveiled many low-frequency alternative isoforms, indicating relatively low splicing fidelity in fission yeast (Bitton et al. 2015;Stepankiw et al. 2015;Kuang et al. 2017). However, regulated alternative splicing has not been observed as a major issue in mitotically active fission yeast cells (Grützmann et al. 2014). Interestingly, certain genes seem to be regulated by splicing during sexual differentiation. ...
Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several life constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter containing the weakest splicing rate with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants (Δcwf12 and Δsaf5), both of them related to the NineTeen Complex (NTC). We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.
... AS is found in a variety of fungal species and is important to the establishment of multicellular complexity [25,57]. This posttranscriptional modification would primarily generate transcript isoforms [58]. ...
Coprinopsis cinerea is one of the model species used in fungal developmental studies. This mushroom-forming Basidiomycetes fungus has several developmental destinies in response to changing environments, with dynamic developmental regulations of the organism. Although the gene expression in C. cinerea development has already been profiled broadly, previous studies have only focused on a specific stage or process of fungal development. A comprehensive perspective across different developmental paths is lacking, and a global view on the dynamic transcriptional regulations in the life cycle and the developmental paths is far from complete. In addition, knowledge on co- and post-transcriptional modifications in this fungus remains rare. In this study, we investigated the transcriptional changes and modifications in C. cinerea during the processes of spore germination, vegetative growth, oidiation, sclerotia formation, and fruiting body formation by inducing different developmental paths of the organism and profiling the transcriptomes using the high-throughput sequencing method. Transition in the identity and abundance of expressed genes drive the physiological and morphological alterations of the organism, including metabolism and multicellularity construction. Moreover, stage- and tissue-specific alternative splicing and RNA editing took place and functioned in C. cinerea. These modifications were negatively correlated to the conservation features of genes and could provide extra plasticity to the transcriptome during fungal development. We suggest that C. cinerea applies different molecular strategies in its developmental regulation, including shifts in expressed gene sets, diversifications of genetic information, and reversible diversifications of RNA molecules. Such features would increase the fungal adaptability in the rapidly changing environment, especially in the transition of developmental programs and the maintenance and balance of genetic and transcriptomic divergence. The multi-layer regulatory network of gene expression serves as the molecular basis of the functioning of developmental regulation.
... The most prevalent types of alternative splicing in fungi are intron retention followed by alternative first and last exons (Keren et al., 2010;Sieber et al., 2018). Overall, the alternative splicing frequency was estimated to be 6.4% of the annotated fungal genes; however, it can vary from 0% in some unicellular Ascomycetes to almost 20% in some Basidiomycetes (Grutzmann et al., 2014). Last, due to the variation in intron properties among fungi, the achieved recall rates indicated a substantial share of introns that were not identified (Figure 4a), especially outside the Dikarya fungi (Ascomycota and Basidiomycota). ...
Metagenomics provides a tool to assess the functional potential of environmental and host-associated microbiomes based on the analysis of environmental DNA: assembly, gene prediction and annotation. While gene prediction is straightforward for most bacterial and archaeal taxa, it has limited applicability in the majority of eukaryotic organisms, including fungi that contain introns in gene coding sequences. As a consequence, eukaryotic genes are underrepresented in metagenomics datasets and our understanding of the contribution of fungi and other eukaryotes to microbiome functioning is limited. Here, we developed a machine intelligence-based algorithm that predicts fungal introns in environmental DNA with reasonable precision and used it to improve the annotation of environmental metagenomes. Intron removal increased the number of predicted genes by up to 9.1% and improved the annotation of several others. The proportion of newly predicted genes increased with the share of eukaryotic genes in the metagenome and-within fungal taxa-increased with the number of introns per gene. Our approach provides a tool named SVMmycointron for improved metagenome annotation, especially of microbiomes with a high proportion of eukaryotes. The scripts described in the paper are made publicly available and can be readily utilized by microbiome researchers analysing metagenomics data.
... First, differences in gene expression [stemming from regulation, epigenetic control or differences in gene copy number], could result in the repression of an Avr protein or in the overproduction of a virulence factor [a protein capable of breaking down plant resistance]. Second, sequence polymorphism and/or structural variation within effector proteins [resulting from mutations or alternative splicing] could hinder host recognition, alter Avr gene transcript abundance or lead to new parasitic functions [20][21][22]. ...
The soybean cyst nematode (Heterodera glycines, SCN), is the most damaging disease of soybean in North America. While management of this pest using resistant soybean is generally still effective, prolonged exposure to cultivars derived from the same source of resistance (PI 88788) has led to the emergence of virulence. Currently, the underlying mechanisms responsible for resistance breakdown remain unknown. In this study, we combined a single nematode transcriptomic profiling approach with long-read sequencing to reannotate the SCN genome. This resulted in the annotation of 1932 novel transcripts and 281 novel gene features. Using a transcript-level quantification approach, we identified eight novel effector candidates overexpressed in PI 88788 virulent nematodes in the late infection stage. Among these were the novel gene Hg-CPZ-1 and a pioneer effector transcript generated through the alternative splicing of the non-effector gene Hetgly21698. While our results demonstrate that alternative splicing in effectors does occur, we found limited evidence of direct involvement in the breakdown of resistance. However, our analysis highlighted a distinct pattern of effector upregulation in response to PI 88788 resistance indicative of a possible adaptation process by SCN to host resistance.
... Transcriptomes allow us to describe alternative splicing complexity of organisms at an unprecedented depth. Several studies looked at alternative splicing in fungi (Zhao et al. 2013, Grützmann et al. 2014, Jin et al. 2017), including Agaricomycetes model systems (Lugones et al. 1999a, Gehrmann et al. 2016. The Basidiomycota and, in particular, the Agaricomycetes displayed a higher percentage of spliced genes than did other groups of fungi (Grützmann et al. 2014. ...
... Several studies looked at alternative splicing in fungi (Zhao et al. 2013, Grützmann et al. 2014, Jin et al. 2017), including Agaricomycetes model systems (Lugones et al. 1999a, Gehrmann et al. 2016. The Basidiomycota and, in particular, the Agaricomycetes displayed a higher percentage of spliced genes than did other groups of fungi (Grützmann et al. 2014. Results of these studies highlighted that, of the several possible splicing events, intron retention (IR) is the most common in fungi while exon-skipping, which most amply contributes to generating protein isoforms, accounts for only 0.5-2.5 % of fungal splicing events. ...
Fruiting bodies (sporocarps, sporophores or basidiomata) of mushroom-forming fungi ( Agaricomycetes ) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates their growth, tissue differentiation and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is still limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim at a comprehensive identification of conserved genes related to fruiting body morphogenesis and distil novel functional hypotheses for functionally poorly characterised ones. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported to be involved in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defence, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1 480 genes of Coprinopsis cinerea , and their orthologs in Agaricus bisporus , Cyclocybe aegerita , Armillaria ostoyae , Auriculariopsis ampla , Laccaria bicolor , Lentinula edodes , Lentinus tigrinus , Mycena kentingensis , Phanerochaete chrysosporium , Pleurotus ostreatus , and Schizophyllum commune , providing functional hypotheses for ~10 % of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes . We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi.
... In Drosophila, the Dscam gene can generate over 38,000 distinct mRNA isoforms [19], which is more transcripts than the total number of genes in this organism (∼14,500). Notably, AS frequency is far less frequent in fungi than in animals, ranging from less than 1% in the budding yeast to 18% in the human pathogen Cryptococcus neoformans [20]. Due to genomic features (few and short introns), intron retention (IR) is the most prevalent splicing type found in fungi (reviewed in [21]). ...
... In our prediction, the proportion of the different patterns of ASEs is in accordance with what has been observed in other filamentous fungi [52]. Regarding the prevalence, we found almost 30% of the genes subjected to AS, which is far more than the 6% found in average for ascomycete fungi [20]. However, this later estimation is based on ESTs and might underestimate the real number of ASEs [21]. ...
Background
Publicly available RNA-seq datasets are often underused although being helpful to improve functional annotation of eukaryotic genomes. This is especially true for filamentous fungi genomes which structure differs from most well annotated yeast genomes. Podospora anserina is a filamentous fungal model, which genome has been sequenced and annotated in 2008. Still, the current annotation lacks information about cis-regulatory elements, including promoters, transcription starting sites and terminators, which are instrumental to integrate epigenomic features into global gene regulation strategies.
Results
Here we took advantage of 37 RNA-seq experiments that were obtained in contrasted developmental and physiological conditions, to complete the functional annotation of P. anserina genome. Out of the 10,800 previously annotated genes, 5’UTR and 3’UTR were defined for 7554, among which, 3328 showed differential transcriptional signal starts and/or transcriptional end sites. In addition, alternative splicing events were detected for 2350 genes, mostly due alternative 3’splice sites and 1732 novel transcriptionally active regions (nTARs) in unannotated regions were identified.
Conclusions
Our study provides a comprehensive genome-wide functional annotation of P. anserina genome, including chromatin features, cis-acting elements such as UTRs, alternative splicing events and transcription of non-coding regions. These new findings will likely improve our understanding of gene regulation strategies in compact genomes, such as those of filamentous fungi. Characterization of alternative transcripts and nTARs paves the way to the discovery of putative new genes, alternative peptides or regulatory non-coding RNAs.
... The most extreme example of AS is the Drosophila melanogaster gene DSCAM, which potentially encodes 38,016 distinct isoforms through AS (Graveley, 2005). AS has likewise been reported in microbial eukaryotes, and the ratio ranges from 0.2% in the non-pathogenic yeast Saccharomyces cerevisiae (Grutzmann et al., 2014) to 24% in the plant-pathogenic oomycete Pseudoperonospora cubensis (Burkhardt et al., 2015), which is much lower than that in plants and animals. ...
... Despite the great progress in related research, AS events and regulation in microbial eukaryotes remain largely elusive, especially in plant pathogens. A genome-wide study of 23 fungal genomes has revealed that pathogenic species have much higher AS rates than nonpathogenic species (Grutzmann et al., 2014). Host-specific and infection-specific AS were observed in S. sclerotiorum and Magnaporthe oryzae (Ibrahim et al., 2021;Jeon et al., 2022), respectively. ...
... Only a few genome-wide AS events have been identified in fungi. It has been reported that the ratio of AS events ranges from 0.2% in the non-pathogenic Saccharomyces cerevisiae to 9.5% in the filamentous fungus Aspergillus niger (Grutzmann et al., 2014) and from 2.3 to 18.2% in human pathogenic fungi (Stepankiw et al., 2015). A recent study showed that 1,487 S. sclerotiorum genes exhibited alternative isoforms on diverse host plants, which may contribute to expanding the host spectrum of S. sclerotiorum (Ibrahim et al., 2021). ...
Alternative splicing (AS) is an important post-transcriptional mechanism promoting the diversity of transcripts and proteins to regulate various life processes in eukaryotes. Sclerotinia stem rot is a major disease of Brassica napus caused by Sclerotinia sclerotiorum, which causes severe yield loss in B. napus production worldwide. Although many transcriptome studies have been carried out on the growth, development, and infection of S. sclerotiorum, the genome-wide AS events of S. sclerotiorum remain poorly understood, particularly at the infection stage. In this study, transcriptome sequencing was performed to systematically explore the genome-scale AS events of S. sclerotiorum at five important infection stages on a susceptible oilseed rape cultivar. A total of 130 genes were predicted to be involved in AS from the S. sclerotiorum genome, among which 98 genes were differentially expressed and may be responsible for AS reprogramming for its successful infection. In addition, 641 differential alternative splicing genes (DASGs) were identified during S. sclerotiorum infection, accounting for 5.76% of all annotated S. sclerotiorum genes, and 71 DASGs were commonly found at all the five infection stages. The most dominant AS type of S. sclerotiorum was found to be retained introns or alternative 3′ splice sites. Furthermore, the resultant AS isoforms of 21 DASGs became pseudogenes, and 60 DASGs encoded different putative proteins with different domains. More importantly, 16 DASGs of S. sclerotiorum were found to have signal peptides and possibly encode putative effectors to facilitate the infection of S. sclerotiorum. Finally, about 69.27% of DASGs were found to be non-differentially expressed genes, indicating that AS serves as another important way to regulate the infection of S. sclerotiorum on plants besides the gene expression level. Taken together, this study provides a genome-wide landscape for the AS of S. sclerotiorum during infection as well as an important resource for further elucidating the pathogenic mechanisms of S. sclerotiorum.
