Intron distribution and emerging role of alternative splicing in fungi

ABSTRACT

Spliceosomal introns are noncoding sequences that are spliced from pre-mRNA. They are ubiquitous in eukaryotic genomes, although the average number of introns per gene varies considerably between different eukaryotic species. Fungi are diverse in terms of intron numbers ranging from 4% to 99% genes with introns. Alternative splicing is one of the most common modes of posttranscriptional regulation in eukaryotes, giving rise to multiple transcripts from a single pre-mRNA and is widespread in metazoans and drives extensive proteome diversity. Earlier, alternative splicing was considered to be rare in fungi, but recently, increasing numbers of studies have revealed that alternative splicing is also widespread in fungi and has been implicated in the regulation of fungal growth and development, protein localization and the improvement of survivability, likely underlying their unique capacity to adapt to changing environmental conditions. However, the role of alternative splicing in pathogenicity and development of drug resistance is only recently gaining attention. In this review, we describe the intronic landscape in fungi. We also present in detail the newly discovered functions of alternative splicing in various cellular processes and outline areas particularly in pathogenesis and clinical drug resistance for future studies that could lead to the development of much needed new therapeutics.

Alternative splicing is as common in fungi as in higher eukaryotes and is used to generate different mRNA isoforms, and the changing isoform ratio with different stresses possibly contributes to the adaptation of fungi to different environmental conditions.

INTRODUCTION

Fungal infections are a common cause of global morbidity and mortality and represent a growing threat to public health particularly due to the increasing numbers of immunocompromised patients (Friedman and Schwartz 2019). The severity of fungal infections varies from superficial to life-threatening systemic infections depending upon the immune status of the host. They are caused by various types of fungi capable of colonizing and invading several human tissues. The predominant pathogens associated with invasive fungal infections are Candida and Cryptococcus species in immunocompromised individuals and, together with Aspergillus, these account for ∼90% of deaths due to fungal diseases (Fisher et al. 2020). The high levels of morbidity and mortality are caused by unique challenges in antifungal drug discovery, including the limited number of specific druggable targets due to pathogenic fungi being eukaryotic organisms. Current antifungal therapies have largely exploited the fungal cell wall and cell membrane components (ergosterol). However, the antifungal repertoire is limited to only very few chemical entities and is being further diminished by recent reports of increasing drug resistance and adverse drug–drug interactions (de Oliveira Santos et al. 2018). Thus, limited antifungal classes, together with the increasing prevalence of bloodstream infections and antifungal multidrug resistance, underscore the critical need for new and more effective antifungals.

Fungi display a remarkable plasticity when grown under harsh environmental conditions. This is a key factor to their general success as pathogens and to their ability to develop resistance to antifungal drugs. The process of alternative splicing (AS) appears to contribute to the adaptation of fungi in different stress conditions. AS occurs in higher eukaryotes and results in the production of multiple isoforms from a single pre-mRNA, thereby massively diversifying the proteome (Liu et al. 2017). AS has been implicated in a wide range of cellular processes, including protein localization, protein diversity, enzymatic activity, apoptosis, changes in ion channel activity and mRNA stability. (Chowdhury et al. 2005; Miyake et al. 2005; Vallejo-Illarramendi, Domercq and Matute 2005; Vegran et al. 2005; Dolzhanskaya, Merz and Denman 2006; Kelemen et al. 2013). The importance of AS in humans, for example, is manifested by numerous diseases that result from aberrant splicing (Kelemen et al. 2013; Anna and Monika 2018). AS represents one of the causes of drug resistance in humans and targeting AS has also led to the discovery of new therapeutics (Tang et al. 2013; Le et al. 2015; Zhao 2019; Roberts, Langer and Wood 2020; Suñé-Pou et al. 2020). However, the impact of AS in fungi remains poorly understudied with only a handful of examples available (Grützmann et al. 2014; Jin et al. 2017). Recent advances in bioinformatics and the availability of several sequenced fungal genomes have revealed that fungi like metazoans contain introns and show huge variation in intron numbers and intron densities; hence, the percentage of genes undergoing AS also varies considerably (Grützmann et al. 2014; Linde et al. 2015; Sieber et al. 2018). In this review, we highlight the functions of introns and the role of AS in various biological processes in fungi and its possible contribution toward virulence and drug resistance in human fungal pathogens.

INTRON LANDSCAPE IN FUNGI

Eukaryotic genes often contain introns (the intervening sequences) that must be spliced out of the gene transcripts for the coding sequences to be fully expressed. There is a large variation in intron numbers between different eukaryotic species, with a much higher prevalence of introns in the genomes of higher eukaryotes. For example, introns comprise ∼25% of the human genome with an average of more than seven introns per gene (Sakharkar et al. 2004; Roy and Gilbert 2006). In yeasts, the intron-containing genes vary considerably between 2.5% and 99%. For example, Candida glabrata has 2.5–4% intron-containing genes, while it is 5%, 6–8%,14% 47% and 99% in Saccharomyces cerevisiae,Candida albicans, Yarrowia lipolytica, Schizosaccharomyces pombe and Cryptococcus neoformans, respectively (Table 1). Intron densities (number of introns per gene) in fungi also vary. For example, in yeast like C. albicans,C. glabrata and S. cerevisiae, the genes mostly contain single introns (Davis et al. 2000; Wood et al. 2002; Neuveglise, Marck and Gaillardin 2011; Janbon et al. 2014; Schreiber et al. 2015), while fungi like Aspergillus fumigatus,Histoplasma capsulatum,Lichtheimia corymbifera and C. neoformans have 54–76% genes with multiple introns (Sieber et al. 2018). In general, fungal genes have comparatively shorter introns and 88–99.8% of the introns have canonical splice sites (5′GU…3′AG) with the noncanonical splice site, 5′GC…3′AG, in 1–2% introns and 5′AU…3′AC in ∼0.09% introns (Kupfer et al. 2004; Grützmann et al. 2014; Sieber et al. 2018).

Table 1.

Total number of introns along with corresponding percentages of genes undergoing AS (or expressing more than one isoform) in different fungal genomes.

Fungi	Number of genes	Number of introns	% of genes undergoing AS
Ascomycota
Arthroderma benhamiae	7984	10 332	8.2
Histoplasma capsulatum	11 216	42 485	38.82
Paracoccidioides brasiliensis	9132	28 179	15.4
Coccidioides immitis	10 440	17 815	13.4
Aspergillus nidulans	9541	16 797	7.3
Aspergillus niger	10 597	17 668	9.5
Aspergillus fumigatus	10 144	30 312	30.21
Neurospora crassa	9841	14 323	8.8
Podospora anserina	10 257	11 261	4.8
Saccharomyces cerevisiae	5781	358	0.18
Candida albicans	6620	867	4.08
Candida glabrata	5632	292	1.95
Schizosaccharomyces pombe	5073	3878	0.6
Trichoderma reesei	9143	18 802	2.5
Trichoderma longibrachiatum	10 792	16 465	48.9
Botryotinia fuckeliana	16 389	22 334	2.7
Gibberella zeae	23 218	38 261	5.9
Magnaporthe grisea	14 010	18 795	7.9
Mycosphaerella graminicola	10 952	17 661	6.1
Phaeosphaeria nodorum	15 983	21 371	2.4
Pichia stipitis	5807	2580	0
Basidiomycota
Cryptococcus neoformans	6604	40 000	59
Coprinopsis cinerea	13 544	30 180	8.6
Laccaria bicolor	18 216	36 757	5.9
Phanerochaete chrysosporium	10 048	48 688	7.7
Ustilago maydis	6522	4279	2.3
Mucoromycotina
Rhizopus oryzae	17 459	40 515	2.3
Lichtheimia corymbifera	11 350	54 131	18.98

The data in Table 1 are taken from references Stajich, Dietrich and Roy 2007; Grützmann et al. 2014; Xie et al. 2015; Gonzalez-Hilarion et al. 2016; and Sieber et al. 2018.

Recent studies have uncovered many roles of introns in fungal genomes (Niu and Yang 2011; Jo and Choi 2015; Bonnet et al. 2017; Shaul 2017; Morgan, Fink and Bartel 2019; Parenteau et al. 2019; Lim et al. 2021). The roles of introns and AS in fungi are summarized in Fig. 1. For example, the physical presence of introns in the S. cerevisiae genome regulates cell survival under starvation conditions by enhancing the repression of ribosomal protein genes in a TORC1-dependent manner (Morgan, Fink and Bartel 2019; Parenteau et al. 2019).

Figure 1.

Schematic representation of intronic regulation of various processes including AS in various fungal species. Introns regulate various processes like nonsense-mediated decay (NMD), genome stabilization and cell growth under nutrient-deficient conditions. Introns also regulate other processes by regulating AS. Through AS, introns further function in fine-tuning gene expression and thus regulating cellular responses to environmental stresses that contribute to pathogenicity (?) and drug resistance (?). AS generates multiple isoforms from a single pre-mRNA molecule that may code for different proteins with different localizations and thus generate proteome diversity. The question marks (?) indicate emerging roles of AS that need further experimental verifications.

Introns also play important roles by harboring noncoding RNA (ncRNA) genes like microRNAs and snoRNAs. For example, a GLC7 intron harbors a ncRNA, wherein its ncRNA region rather than the intron regulates cell growth under salt stress in S. cerevisiae (Jo and Choi 2015; Hooks et al. 2016). Recently, two genes, NOG2 and RPL7B, encoding a putative GTPase-associated pre-60S ribosomal subunit and a 60S ribosomal protein, respectively, were found to have a conserved intron position in fungi, and the ncRNA, snR191, was found to be embedded inside the intron of NOG2 and snR59 in the intron of RPL7B (Lim et al. 2021). The introns of two other genes, RPS22B and RPL18A, form a double-stranded stem loop structure recognized by the yeast RNaseIII ortholog, Rnt1p, that triggers the degradation of these transcripts by Xrn1p and Rat1p (Danin-Kreiselman, Lee and Chanfreau 2003). These introns are now predicted to contain ncRNAs (Hooks et al. 2016).

SPLICING AND AS IN FUNGI

AS is a complex, regulatory process by which multiple transcripts are generated from a single pre-mRNA, which expands proteomic diversity. Introns regulate AS by harboring specific sequences called splice sites, which are recognized by the splicing machinery or spliceosome, shown in Fig. 2. The observed frequency of AS events is very low (<10%) in yeast as compared with plants (∼60%) and animals (>90% in humans) (Kempken 2013; Sablok et al. 2017). A study conducted by Grutzmann et al. in 2014 on 27 different fungi reports an average AS rate (percentage of gene undergoing AS) of 8.6% in Basidiomycota and 7.2% in Ascomycota (Grützmann et al. 2014). However, recent studies report that AS occurs at a frequency much higher than reported earlier (Table 1). For example, in Trichoderma longibrachiatum, an AS rate of 48.9% is reported (Xie et al. 2015) and in C. neoformans, an AS rate of 59% is reported (Gonzalez-Hilarion et al. 2016). AS events can be split into different types depending on the splice sites used and the fate of the introns and exons in the final mRNA (Fig. 3). The most common type of AS events in fungi is intron retention (IR) type (McGuire et al. 2008; Grützmann et al. 2014).

Figure 2.

Schematic representing the structure of an intron-containing gene with elements regulating AS.(A) Structure of intron-containing genes with exons represented as boxes and intron as a straight line. The intron containing sequences called splice sites are recognized by the splicing machinery (spliceosome) that help in the excision of intron and subsequent ligation of exons. More than 98% introns have canonical splice sites (5′ splice site, 3′ splice site and branch point) as shown in the diagram. (B) Apart from the splice sites, the introns and exons also contain sequences that help in either enhancing or repressing the pre-mRNA splicing by recruiting specific trans-regulatory elements. The serine–arginine (SR) proteins bind to the splicing enhancer (ISE/ESE) sequences (shown in green color) and activate splicing by allowing U1 and U2 snRNPs to bind to 5′SS and branch point, respectively, and subsequent assembly of the spliceosome. On the other hand, hnRNPs when bound to splicing silencer (ISS/ESS) sequences (shown in grey color) repress splicing by blocking the spliceosome assembly. ISE, intronic splicing enhancer; ESE, exonic splicing enhancer; ISS, intronic splicing silencer; ESS, exonic splicing silencer; SF, splicing factors.

Figure 3.

A schematic diagram showing the different types of AS events in fungi. In the cassette exon, the exon is either retained or spliced out. In the IR type, the intron is either retained or spliced out, a common type occurring in fungi. In mutually exclusive AS, the two internal exons (represented by orange and green boxes) never appear together in the final product after splicing. If one is retained, the other is spliced out and vice versa. The spliceosome can also use alternative 3′ or 5′ splice sites and alternative promoters and alternative poly A sites to generate diverse isoforms from a single pre-mRNA. The exons are represented by boxes and introns by a straight line.

EMERGING ROLES OF AS IN FUNGI

In contrast to humans, where alternate splicing events have been extensively studied, the biological impact of AS in fungi remains relatively unexplored. However, studies are emerging that associate AS in fungi with diverse functions including in environmental adaptation, protein localization, meiosis and gene expression, which are described in detail later.

AS in growth and development

AS is used during growth and development in fungi. For example, a pheromone receptor gene, PRE-1 (with three exons and two introns), undergoes AS in Neurospora crassa to produce two splice variants due to the retention of its last intron. The IR leads to the loss of 322 amino acids due to the introduction of a premature termination codon (PTC) from the C-terminus of the protein, resulting in the loss of six ubiquitination sites. The two isoforms coexist in vegetative and reproductive mycelia (Strandberg et al. 2013). Another gene in N. crassa, TOB55, which encodes a major component of the topogenesis of mitochondrial outer membrane β-barrel proteins (TOB) complex and is essential for the cell viability, undergoes AS. The TOB55 gene is multiexonic because it has three introns and four exons. The presence of alternative splice sites in the second exon results in the AS of the TOB55 gene yielding three confirmed protein isoforms. Neurospora crassa strains expressing only the long form of the Tob55 protein exhibited growth defects at higher temperatures (Hoppins et al. 2007).

AS influences protein localization

AS can also impact the subcellular localization of proteins that are encoded by genes containing a functional intron. Retention of these introns in the mRNA introduces specific target signal sequences in the final protein, resulting in differential localization of the protein. There are several examples of AS controlling protein localization in S. cerevisiae. First, a serine/threonine protein phosphatase type 2C (PP2C) encoding gene PTC7 in S. cerevisiae undergoes AS to produce two isoforms, where the intron is spliced out in one of the isoforms and is retained in the other. Due to the presence of a functional intron, both the isoforms code for different proteins which localize to different cellular compartments. The protein coded from the spliced isoform localizes to mitochondria and the protein from the unspliced isoform localizes to the nuclear envelope (Juneau, Nislow and Davis 2009). Another gene in S. cerevisiae,FES1, encodes a Hsp70 nucleotide exchange factor and is essential for protein quality control. FES1 undergoes AS to produce two isoforms which code for two different proteins (Fes1S and Fes1L) with alternative C termini. Fes1L localizes to the nucleus and functions as a Hsp70 nucleotide exchange factor, while Fes1S is confined to the cytosol where it maintains proteostasis. AS of the FES1 transcript is also modulated by heat shock (Gowda et al. 2016).

The regulation of protein localization by AS has also been discovered in several other fungi. For example, in Y. lipolytica, AS governs the cellular localization of a central carbon metabolism enzyme malate dehydrogenase encoded by the ylMDH2 gene. The ylMDH2 gene has three exons and two introns, and the second intron possesses two 3′ splice sites. The alternate use of these splice sites generates two isoforms with cytosolic and peroxisomal localization (Kabran et al. 2012). Also, in Aspergillus nidulans and Ustilago maydis, AS affects protein localization of core glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK) to the cytosol or peroxisomes. In U. maydis, the GAPDH gene has two 5′ splice sites and alternative polyA sites while the PGK1 gene has an additional stop codon. To generate the peroxisomal target signal type 1 (PTS1) in these proteins, U. maydis uses the alternate 5′ splice sites in GAPDH and the alternate stop codon by ribosomal read-through in the case of PGK1. However, compared with U. maydis, A. nidulans uses exactly the opposite mechanisms. Specifically, peroxisomal GAPDH is generated by ribosomal readthrough, while peroxisomal PGK is generated by AS (Freitag, Ast and Bölker 2012). The dual targeting of glycolytic enzymes (GAPDH and PGK) due to AS also occurs in some other fungi such as in N. crassa,Botrytis cinerea,Y. lipolytica,Sphacelotheca reilianum,Penicillium chrysogenum and Aspergillus flavus (Freitag, Ast and Bölker 2012). In C. albicans, AS events in a pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (GND1), generates its two isoforms which code for proteins localizing to either the cytosol or peroxisomes. The intron in GND1 encodes a peroxisomal target signal type 2 (PTS2), which leads to the peroxisomal localization of the alternatively spliced isoform while the spliced isoform, lacking PTS2, localizes to the cytosol (Strijbis et al. 2012).

AS impacts meiosis, histone modification and gene expression

In metazoans, during meiosis, cells undergo dramatic reprogramming, not only at the gene transcriptional level, but also at the posttranscriptional level, including substantial changes in AS events (Schmid et al. 2013). Fungi also employ modulations in AS events during meiosis despite having a comparatively streamlined genome as compared with metazoans. A critical meiotic splicing regulator in S. cerevisiae, Mer1, regulates the splicing of four meiotic pre-mRNAs (MER2,MER3,AMA1 and SPO22) (Qiu, Schwer and Shuman 2011; Venkataramanan et al. 2017). In S. cerevisiae, MER2 and MER3 genes are essential for meiosis and both the genes possess an intron with noncanonical 5′ splice sites. MER2 is transcribed in both mitosis and meiosis, but it is efficiently spliced only during meiosis when it generates a functional product. The splicing of MER2 is dependent on MER1, which is transcribed only during meiosis. MER3 is also transcribed and spliced only during meiosis (Engebrecht, Voelkel-Meiman and Roeder 1991; Nakagawa and Ogawa 1999).

AS has also been associated with histone modification in fungi. The Gcn5 gene is a paradigm histone acetyl transferase (HAT) that is involved in morphogenesis and virulence of C. albicans (Shivarathri et al. 2019). The S. cerevisiae GCN5 genetically interacts with MSL1 and LEA1, encoding components of U2snRNP, which is important for branch site recognition in pre-mRNA splicing. The Gcn5 HAT activity helps mediate co-transcriptional recruitment and assembly of spliceosomes and recognition of the branch point. Additionally, the deletion of histone deacetylases Hos3 and Hos2 results in persistent association of U2 snRNP with branchpoints, thus, interfering with the spliceosome rearrangements (Gunderson and Johnson 2009; Gunderson, Merkhofer and Johnson 2011). Another chromodomain-containing protein, Eaf3, mediates the interaction between H3K36 methylation and the splicing machinery, and the loss of Eaf3 leads to defective splicing (Leung et al. 2019). The Swi/Snf complex, a chromatin remodeling complex, also regulates splicing as mentioned above. Together, these highlight that histone modifications and spliceosome assembly are tightly linked and any change in transcription could affect the pre-mRNA splicing (Johnson and Vilardell 2012; Aslanzadeh et al. 2018).

The role of AS in fine-tuning the gene expression is illustrated by observing the effect of intron deletion in ribosomal protein genes (RPGs). The deletion of all the introns of RPGs revealed that 84% of the deleted introns affect the expression of its host genes (Parenteau et al. 2011). Moreover, 33% of the duplicated RPGs are regulated in an intron-dependent manner and the deletion of introns in one gene affects the expression of its other copy (Niu and Yang 2011; Parenteau et al. 2011; Shaul 2017). Furthermore, a comparative analysis of the RNA seq data from S. cerevisiae,C. albicans,S. pombeandN. crassa revealed that mRNA transcribed from intron-containing genes have higher expression levels than from non-intronic genes, again demonstrating a role for introns in enhancing gene expression (Lim et al. 2021). In addition, 5′ UTR introns enhance the translation efficiency in S. cerevisiae, the mechanism remains unknown (Hoshida et al. 2017). Other examples include genes like SUS1,YRA1 and RPL30 in S. cerevisiae where the accumulation of the final protein product results in the splicing inhibition of its own transcript. The SUS1 gene is a component of the SAGA complex and plays a role in both transcription and mRNA export (Pascual-García and Rodríguez-Navarro 2009; Hossain, Rodriguez and Johnson 2011). This gene is having two introns. Of note, only nine genes are reported to have multiple introns in S. cerevisiae (Mitrovich et al. 2007). The physical presence of both introns in SUS1 is required for its efficient splicing and removal of one intron affects the splicing of the other intron (Cuenca-Bono et al. 2011). The first intron has a noncanonical 5′ splice site and branchpoint sequence that decreases its splicing and enhances the chances of its retention. Both isoforms generated by AS are essential for temperature sensitivity and H2B histone deubiquitination (Hossain, Rodriguez and Johnson 2011). The expression of the Sus1 protein is regulated by the efficient splicing and decay of its transcript (Cuenca-Bono et al. 2011). The YRA1 gene is required for the export of polyA mRNAs from the nucleus, connecting transcription to mRNA export. Overexpression of Yra1p arrests the cell growth, therefore, maintaining the appropriate level of Yra1p is critical for cell viability. Yra1p is autoregulated by a negative feedback loop. When the expression of Yra1p increases, it binds to its intron-containing transcript (pre-mRNA) and inhibits splicing, thereby decreasing the level of Yra1p (Preker, Kim and Guthrie 2002; Preker and Guthrie 2006). RPL30 is another example of regulated splicing. RPL30 encodes a ribosomal protein of the large subunit and is involved in pre-rRNA processing. When the Rpl30 protein accumulates in the cell, it binds to the RPL30 transcript and inhibits its splicing by preventing spliceosomal assembly (Vilardell and Warner 1994; Vilardell, Yu and Warner 2000; Bragulat et al. 2010).

AS in pathogenesis and virulence

The first clue of AS involvement in pathogenicity came from a study that reported an average AS rate in human pathogenic fungi to be double the AS rates of nonpathogenic fungi (Grützmann et al. 2014). However, there are not many experimentally validated examples establishing roles of AS events in fungal pathogenesis. Nonetheless, different genes that play a role in pathogenesis in fungi have been predicted to undergo AS. For example, in the encapsulated yeast C. neoformans glucuronoxylomannan is a capsular polysaccharide. The C. neoformans genes CNBB3380 and CNBA6810; involved in the production and modification of glucuronoxylomannan, are affected by AS (Grützmann et al. 2014) and the structural changes in glucuronoxylomannan during morphological switching helps in the evasion of the host immune system and hence contributes to the pathogenicity of C. neoformans (Jain and Fries 2008; Grützmann et al. 2014; Denham and Brown 2018; Zaragoza 2019). Another gene in C. neoformans,CIN1 (cryptococcal intersectin1), similar to human intersectin ISTN1, is a multidomain adaptor protein required for growth, intracellular transport and the formation of virulence factors like the capsule, urease, phospholipase B and melanin production. The gene undergoes AS to form two Cin1p isoforms, just like human ISTN1, longer Cin1-L and shorter Cin1-S due to the presence of an alternative 3′ splice site in its intron 6. However, the function of these isoforms is unknown (Shen et al. 2010, 2012).

In a plant fungal pathogen, U. maydis, the UmRRM75 gene encoding a protein with three RNA recognition motifs, affects the morphogenesis and virulence of this pathogen. This gene is multiexonic with four introns and five exons, and undergoes AS due to the presence of an alternative 3′ splice site in its third exon (Rodríguez-Kessler et al. 2012). Furthermore, the important carbon metabolism enzymes GAPDHandPGK have peroxisomal localization due to AS (discussed earlier), and cells lacking in the peroxisomal isoforms of these genes show reduced virulence in U. maydis (Freitag, Ast and Bölker 2012). Also, other factors that contribute to the pathogenicity of fungi including adaptation to environmental stresses and different genes involved in stress responses, such as TPS1 (trehalose-6-phosphate synthase), HSP30 (heat shock protein) and DDR48 (DNA damage responsive), are also affected by AS in various fungi reported by Grutzmann et al. (2014).

The specificity of pre-mRNA requires a proper recruitment of splicing factors and any aberration in splicing factor recruitment can be detrimental to the overall physiology of an organism. For example, in fungi, modulation of AS in apoptosis-related genes impacts pathogenesis. In S. cerevisiae, a type 1 arginine methyl transferase, Hmt1, methylates Snp1 (U1 snRNP-specific protein) and regulates the co-transcriptional recruitment of various splicing factors (Chen et al. 2010). Recently, the HMT1 gene was shown to influence the pathogenicity of a plant fungal pathogen M. oryzae wherein its gene MoHMT1, which regulates light and nitrogen starvation-induced autophagy, also regulates genome wide AS events and pathogenicity (Li et al. 2020).

Apart from general splicing events, some of the splicing factors are also shown to influence fungal pathogenicity. For example, a SR-like protein, SLR1, in C. albicans influences filamentation and virulence (Ariyachet et al. 2013). Although the role of SLR1 as a splicing factor is not confirmed in C. albicans, its orthologs in A. nidulans (SWOK) and S. pombe (SRP1) are associated with mRNA processing (Shaw and Upadhyay 2005; Lipp et al. 2015). The glycine-rich protein of M. oryzae, MoGrp1, has an N-terminal RNA recognition motif (RRM) and a C-terminal glycine rich domain with RGG (arginine–glycine–glycine) repeats. It acts as a splicing factor and also regulates fungal virulence by regulating the splicing of the genes MoRAD6 and MST7 involved in infection-related morphogenesis (Gao et al. 2019). From RNA-seq analysis of human pathogenic fungi, it was revealed that the splicing efficiency in response to host conditions is very low, which could be due to the intron retention type of AS (Sieber et al. 2018). Also, the genes that are normally affected by AS in response to host conditions show enrichment in membrane functions, implying that AS may have a role during host invasion. However, these are not yet proven experimentally (Sieber et al. 2018).

AS in stress response

AS in fungi is regulated by various environmental factors, including nutrient signaling and environmental pH changes, which provide them with a remarkable adaptability to withstand environmental stresses (Pleiss et al.2007a,b; Grützmann et al. 2014; Gonzalez-Hilarion et al. 2016). Emerging evidence suggests that AS plays an active role in coping with the cellular stresses of fungi. For instance, the adhesin EPA6 (CAGL0C00110g) and EPA20 (CAGL0E275g) genes in C. glabrata undergo AS and express different isoforms under nitrosative stress and pH change while the EPA3 (CAGL0E006688g) gene expresses different isoforms under nitrosative stress and nutrient rich conditions (Linde et al. 2015). The C. albicans genes RPL30 (ribosomal protein L30) and SPR28 also undergo AS in response to the external environment. RPL30 has a canonical 5′ splice site in the center of its intron and expresses three isoforms in response to temperature changes. One of the seven genes in C. albicans that encode septin proteins, SPR28, contains two introns. The first intron has a noncanonical branch site while the second intron contains a noncanonical 5′ splice site and undergoes AS in response to the α-factor mating pheromone (Mitrovich et al. 2007). The exposure of C. albicans cells to α-factor pheromone results in the differential expression of the splicing factors, LEA1 and SLU7 (Mitrovich et al. 2007). The SOD3 and CAN3 genes in C. albicans undergo AS in response to oxidative stress and acetic acid treatment, respectively (Sieber et al. 2018).

In S. cerevisiae, the splicing of ribosomal protein genes (RPGs) is reported to be influenced by environmental conditions such as the depletion of amino acids or ethanol toxicity (Pleiss et al. 2007b). In C. neoformans, CNAG_06101 (which encodes an ADP/ATP carrier protein), contains an intron in its 5′ UTR that is retained under high temperature stress. In a thermophilic fungus, L. corymbifera, LCOR_03517.1, which encodes a protein with transmembrane activity, undergoes AS with three of its introns being retained under hypoxic conditions. Another gene, LCOR_10856.1, which encodes a sugar transporter also undergoes AS due to the presence of an alternative start site after the first two exons (Sieber et al. 2018). The AFUB_043270 gene, which encodes a transcription factor in A. fumigatus, undergoes AS under a low oxygen environment, with intron retention being more common in control conditions as compared with stress conditions (Sieber et al. 2018).

AS in drug resistance

AS plays an important role in developing resistance to anticancer drugs in humans and hence compounds that can modulate AS are potential therapeutic targets (Siegfried and Karni 2018; Wang and Lee 2018). However, how AS impacts clinical drug resistance in fungi remains underexplored. There are a few instances where an impact of drugs on AS has been demonstrated. For example, in Trichophyton rubrum, several of its genes such as IMPDH,PAKA/STE20 (TERG_03042) and HSP7- undergo the IR type of AS upon exposure to antifungal agents like UDA (undecanoic acid) and TRB (terbinafine) (Neves-da-Rocha et al. 2019). The PAKA/STE20 encoding protein kinase involved in MAPK signal transduction shows retention of intron1 upon exposure to UDA. This IR event generates a premature stop codon and a new start codon immediately, thus generating two isoforms, one full and the other devoid of an essential regulatory CRIB domain. The PAKA kinase, without a regulatory domain, is likely needed to overcome the effects of the antifungal agent UDA (Gomes et al. 2018). PAKA may also be critical for virulence in T. rubrum as in the case of C. albicans where the Ste20-like protein encoded by CST20 gene is required for virulence and hyphal growth (Leberer et al. 1997). Two heat shock protein (HSP)-encoding genes, HSP7-like (TERG_03206) and HSP75-like (TERG_01883), also show IR events upon UDA exposure. These AS events disrupt the ORFs with a premature stop codon and lead to truncated or nonfunctional proteins. The modulation of AS in HSP-encoding genes may be beneficial for cell adaptation to various stresses (Neves-da-Rocha et al. 2019). The PGM (phosphoglucomutase) gene also undergoes AS (by exon skipping) in T. rubrum in response to UDA exposure (Mendes et al. 2018). The AFUB_001340 gene in A. fumigatus, which encodes a putative splicing factor, undergoes AS upon antifungal caspofungin treatment (Sieber et al. 2018). The exposure of N. crassa to antifungal drugs (amphotericin B and ketoconazole) modulates pre-mRNA splicing and results in the IR events in genes that encode asparagine synthetase (asn-2), farnesyltransferase (ram-1) and C6 zinc finger regulator (fluffy). The IR event in these genes is also regulated by inorganic phosphate and pH changes (Mendes et al. 2016).

A recent study by Muzafar et al. explored AS roles in drug resistance in C. albicans and identified several genes that undergo differential AS between the azole-susceptible and azole-resistant isogenic clinical isolates of C. albicans (Muzafar et al. 2020). By using SOD3, a superoxide dismutase encoding gene, as an example, the study showed that AS has a critical role in drug resistance. The expression of individual isoforms of the SOD3 gene in the sod3 null mutant background revealed an interesting role of menadione and amphotericin B on its splicing. The study showed that unlike amphotericin B, menadione inhibits SOD3 splicing and acts as a splicing inhibitor. Menadione exposure resulted in an increased level of unspliced SOD3 isoform that is unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. This splicing inhibitory effect was not seen in the case of amphotericin B which, on the contrary, increased the splicing of SOD3 gene. These observations suggest that AS represents a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans. However, questions such as which factors lead to these splicing modulations still remain unanswered (Muzafar et al. 2020). Table 2 lists the experimentally verified reports of AS in different fungi.

Table 2.

List of genes that undergo AS in different fungi.

Fungi	AS genes	References
Cryptococcus neoformans	CNA07120	(Gonzalez-Hilarion et al. 2016)
	CND00620	(Gonzalez-Hilarion et al. 2016)
	CNA02210	(Gonzalez-Hilarion et al. 2016)
	CNH03510	(Gonzalez-Hilarion et al. 2016)
	CNBB3380	(Jain and Fries 2008; Grützmann et al. 2014)
	CNBA6810	(Jain and Fries 2008; Grützmann et al. 2014)
	CNAG_06101	(Jain and Fries 2008; Grützmann et al. 2014)
	CAS3	(Gonzalez-Hilarion et al. 2016)
	YRA1	(Gonzalez-Hilarion et al. 2016)
	URA4	(Gonzalez-Hilarion et al. 2016)
	ELO3	(Gonzalez-Hilarion et al. 2016)
Fusarium graminearum	FGSG_05122	(Zhao et al. 2013)
	FGSG_04141	(Zhao et al. 2013)
	FGSG_06760	(Zhao et al. 2013)
Neurospora crassa	TOB55	(Hoppins et al. 2007)
	pre-1	(Strandberg et al. 2013)
	asn-2	(Mendes et al. 2016)
	ram-1	(Mendes et al. 2016)
	fluffy	(Mendes et al. 2016)
	hex1	(Leal et al. 2009)
Saccharomyces cerevisiae	PTC7	(Juneau, Nislow and Davis 2009)
	SRC1	(Grund et al. 2008)
	MER2	(Engebrecht, Voelkel-Meiman and Roeder 1991; Nakagawa and Ogawa 1999)
	MER3	(Engebrecht, Voelkel-Meiman and Roeder 1991; Nakagawa and Ogawa 1999)
	SUS1	(Hossain, Rodriguez and Johnson 2011)
	YRA1	(Preker, Kim and Guthrie 2002)
	RPL30	(Vilardell and Warner 1994; Vilardell, Yu and Warner 2000)
	Hmt1	(Chen et al. 2010)
	FES1	(Gowda et al. 2016; Aslanzadeh et al. 2018)
	TAD3	(Aslanzadeh et al. 2018)
Yarrowia lipolytica	ylMDH2	(Kabran et al. 2012)
Ustilago maydis	GAPDH	(Freitag, Ast and Bölker 2012)
	UmRrm75	(Rodríguez-Piña et al. 2019)
Aspergillus nidulans	PacC	(Trevisan et al. 2011)
	PalB	(Trevisan et al. 2011)
	PGK	(Freitag, Ast and Bölker 2012)
Candida albicans	GND1	(Strijbis et al. 2012)
	SOD3	(Sieber et al. 2018; Muzafar et al. 2020)
	CAN3	(Sieber et al. 2018)
	RPL30	(Mitrovich et al. 2007)
	PRE6	(Muzafar et al. 2020)
	INO4	(Muzafar et al. 2020)
	PHA2	(Muzafar et al. 2020)
	AUT7	(Muzafar et al. 2020)
	TRI1	(Muzafar et al. 2020)
	ROB1	(Muzafar et al. 2020)
	MTLA1	(Muzafar et al. 2020)
	CR_06850C_A	(Muzafar et al. 2020)
	C2_00570W_A	(Muzafar et al. 2020)
	C1_00910W_A	(Muzafar et al. 2020)
	CR_03310C_A	(Muzafar et al. 2020)
	C1_10750C_A	(Muzafar et al. 2020)
	SMX2	(Muzafar et al. 2020)
	SPR28	(Mitrovich et al. 2007)
	SKI7/HBS1	(Marshall et al. 2013)
	HAC1	(Sircaik et al. 2021)
Lachancea kluyveri	SKI7/HBS1	(Marshall et al. 2013)
Magnaporthe oryzae	MoHMT1	(Li et al. 2020)
	MoRAD6	(Gao et al. 2019)
	MST7	(Gao et al. 2019)
Candida glabrata	EPA6	(Linde et al. 2015)
	EPA20	(Linde et al. 2015)
	EPA3	(Linde et al. 2015)
Lichtheimia corymbifera	LCOR_03517.1	(Sieber et al. 2018)
Aspergillus fumigatus	AFUB_043270	(Sieber et al. 2018)
	AFUB_001340	(Sieber et al. 2018)
Trichophyton rubrum	impdh	(Gomes et al. 2018)
	pakA/ste20	(Gomes et al. 2018)
	hsp7-like	(Gomes et al. 2018)
	Pgm	(Mendes et al. 2018)
Monascus pilosus	MpLaeA	(Zhang and Miyake 2009)
Schizophyllum commune	G2634198	(Gehrmann et al. 2016)
	G2629174	(Gehrmann et al. 2016)
	G2502024	(Gehrmann et al. 2016)
	G2707155	(Gehrmann et al. 2016)
Shiraia bambusicola	sbFLO	(Liu et al. 2020)
	sbPKS	(Liu et al. 2020)
	sbMFS	(Liu et al. 2020)

CONCLUSIONS AND FUTURE DIRECTIONS

Compared with metazoans, fungal genomes show dramatic variation in intron numbers (Grützmann et al. 2014; Sieber et al. 2018). The intron removal is catalyzed by a complex and dynamic molecular machinery called the spliceosome in a highly coordinated manner, offering the cells an opportunity to regulate gene expression in varied environmental conditions (Pleiss et al. 2007b; Fica and Nagai 2017; Mendoza-Ochoa et al. 2019). Unlike metazoans, AS in fungi does not create much proteome diversity, but rather controls mRNA expression. In fungi, the intron removal is governed by intron definition, in contrast to exon definition used in humans. Notably, IR is the most common strategy in fungi and thus AS differs in terms of regulation from the higher eukaryotes. There are considerable predictions that highlight the frequency of AS in fungi, which require experimental validations (Gallegos and Rose 2015; Monteuuis et al. 2019). The relevance of AS in regulating drug resistance and pathogenesis is emerging and further investigations on the characterization of fungal intronome and AS are required to elucidate how the splicing network responds to cellular stresses, antifungal drugs and host immune response. In future studies, it would be of interest to determine whether AS promotes adaptive advantages or immune evasion by commensal pathogens. Of particular interest, it would be exciting to determine whether AS occurs preferentially on known virulence or drug resistance genes. Therefore, it is expected that the increasing availability of genomic and transcriptomic data and improved bioinformatics tools will yield important advances in discovering novel and potentially disease-relevant roles of AS in pathogenic fungi.

FUNDING

Research in the NC laboratory is supported by a grant from the National Institutes of Health (NIH) (R01AI124499). RP and RDS acknowledge support of a grant from the Department of Science and Technology (DST/EMR/2017/001907). SM acknowledges the Senior Research Fellowship award by the Indian Council of Medical Research (ICMR).

Conflict of interest

None declared.