1 Introduction

Alternative splicing (AS) is a common process which generates more than one RNA transcript from an intron containing gene in eukaryotic organisms. AS plays important biological roles in regulation of biological development and adaptions to the changing environments through increasing both the diversities of transcriptome and proteome (Chaudhary et al., 2019). It is estimated more than 90% genes in humans and ~65-70% genes in plants, such as Arabidopsis and tomato, are subject to alternative splicing (Pan et al., 2008; Zhang et al., 2017; Clark et al., 2019). A recent survey reveals AS events in fungi ranged from 0.2% in non-pathogenic yeast Saccharomyces cerevisiae to 38.44% of expressed transcripts in Shiraia bamlusicola, a parasitic fungus on bamboo twigs (Fang et al.; 2020; Liu et al., 2020).

Aspergillus niger, a filamentous fungal species, is widely used in fermentation industry for producing citric acid, glucoamylase and some other enzymes (Cairns et al., 2018). Identifying alternatively spliced genes in this species may help to improve industrial strains for enzyme production. Glucoamylase mRNA transcripts in A. niger were among the earliest AS cases reported in fungi (Boel et al., 1984). One 169 bp intron was involved in differential mRNA processing leading to two different glucoamylase enzymes G1 and G2 (Boel et al., 1984). Assembling expressed sequence tags (ESTs) identified 56 alternatively spliced genes including glucoamylase genes in A. niger (Semova et al., 2006). Mapping mRNA and EST sequences with spliced transcript-genome alignments further revealed 9.5% AS rate in A. niger (Grützmann et al., 2014). In last ten years RNA sequencing (RNA-seq) technology has been widely used to quantify RNA transcripts of a transcriptome as well as to identify AS events. A number of RNA-seq experiments have been reported in A. niger, such as, Xu et al. (2024) identified a total of 23 out of the 56 lignocellulose-degrading enzyme genes which had AS events with intron retention as the main type of AS events. Clearly, there is a lack of systematic genome-wide identification of alternatively spliced genes in this important species. In considering the importance of the organism in industrial applications, we carry out a systematic genome-wide identification and analysis of AS events in A. niger by integrating available RNA transcripts with RNA-seq data from multiple published projects. The aim is to generate a catalog of genes subjecting to AS in A. niger. Such a collection of these genes with their respective transcript isoform annotation information may serve as a foundation for further characterizing the biological functions and regulations of these genes in this important fungal species for the fermentation industry.

2 Materials and Methods

2.1 Genome, mRNA sequences, and RNA-seq datasets

A. niger reference genome sequences with annotation GFF (Gene feature format) file (CBS 513.88, assembly ASM285v2) and other related files were downloaded from the genome database of the National Center for Biotechnology Infomation (NCBI, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000002855.4/) (Pel et al., 2007). A total of 78,361 A. niger mRNA sequences which includes 46,938 ESTs were downloaded from NCBI nucleotide database. The RNA-seq data was down-loaded from the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra/docs/sradownload/) using SRA Toolkit. The RNA-seq datasets were selected from seven projects which were recently published in eight RNA-seq publications (Table 1). A total of 303 RNA-seq samples generated from diverse treatments were collected (Table 1). The data from the project PRJNA250529 were generated and analyzed by Daly et al. (2017) and van Munster et al. (2020). Daly et al. (2017) investigated the responses of A. niger to ionic liquid (IL) or hydrothermally (HT) pretreated knife-milled wheat straw (KMS) over a time course using RNA-seq and proteomics. van Munster et al. (2020) further analyzed the responses of A. niger to the feedstock Miscanthus and compared the results on wheat straw. Other data were collected including A. niger cultured in peanut or cashew nut flour- based media (Mattison et al., 2021), in steam-exploded sugarcane bagasse (Borin et al., 2017), in sugar beet pulp (Garrigues et al., 2022), in sucrose or inulin (Kun et al., 2023), in glucose or wheat straw (Xu et al., 2024), and wildtype and different deletion strains cultured in glucose (van Leeuwe et al., 2020). We also tested RNA-seq datasets reported in project PRJNA316878 and PRJNA148183 and found the RNA-seq data mapping rate <50%, those data were not included for further analysis.

Table 1 RNA-seq data sources and related reference Note: * The accession numbers of the data can be found in related references and in supplementary files

2.2 mRNA sequence mapping, RNA-seq reads mapping, and AS identification

The procedure for mRNA sequences cleaning and further assembling into a non-redundant set of unique transcripts were described in our previous work (Clark et al., 2019). The final cleaned transcripts consisting of 78,194 sequences were further assembled into a non-redundant set of 23,853 sequences. The assembled nucleotide sequences were mapped to A. niger genome sequences using cutoff values of a minimum 95% identity and >75% length coverage using ASFinder and Sim4 programs (Florea et al., 1998; Min, 2013).

The RNA-seq reads were mapped to the reference genome sequences using TopHat (v2.2.6) with default parameters (Kim et al., 2013). TopHat2 is designed to handle a relatively low error rate, typically considered around 1-2% for most RNA-Seq data, allowing it to accurately map reads even with minor sequencing errors present in the data. The transcript alignment bam file together with annotation GFF file were used as input for Cufflinks (v2.2.1) (Trapnell et al., 2010). The transcript GTF (Gene Transfer Format) files generated from each RNA-seq dataset after running Cufflinks were merged using cuffmerge script within the Cufflinks package for each project. The GTF file generated from merged RNA-seq GTF files in each RNA-seq project was further merged using Cuffcompare script. Astalavista was used for AS event classification (Foissac and Sammeth, 2007). AS events are generally classified as exon skipping (ExonS), alternative donor site (AltD), alternative accepter site (AltA), intron retention (IntronR) and complex event. The complex AS events were counted when two or more basic events occurred in comparing a pair of isoforms. RNA-seq data processing was carried out in our facility and in the Ohio Supercomputer Center.

2.3 Functional annotation of transcripts and data availability

The transcript sequences were retrieved using gtf_to_fasta tool in the TopHat package based on the GTF file generated by Cuffcompare program after merging all GTF files. These transcripts were functionally annotated, including open reading frame (ORF) prediction, BLASTX against UniProt-Swiss-Prot database, protein family (Pfam), and comparison with reference gene models (Min et al., 2005a; Min et al., 2005b). The transcripts sequences, detailed information of AS events, and supplementary files are available at our bioinformatics site (http://bioinformatics.ysu.edu/publication/data/Aniger/).

3 Results

3.1 Mapping mRNA assembled transcripts and RNA-seq data to the genome

Beginning with a total of 78,361 mRNA sequences, after going through the cleaning procedure including trimming poly(A/T) ends and removing contaminants and repetitive sequences, we obtained 78,194 sequences that were further assembled into a non-redundant set of 23,853 transcripts for genome mapping. A total of 19,571 (82.0%) assembled transcripts were mapped to the reference genome.

We mapped a total of 303 RNA-seq datasets to A. niger reference genome (Table 2). The accession numbers and detailed mapping information of these RNA-seq data can be found in a supplementary file (supplementary Table 1). The mapping rates varied from 70.2% to 90.0% with 0.4% to 4.0% reads being mapped to more than one location in the datasets collected from different projects. In total 12.7 billion reads were collected with 10.3 billion reads (~81.0%) being mapped to the genome. Among the mapped reads, 2.7% reads (~0.35 billion) were mapped to two or more genomic loci (Table 2).

Table 2 Mapping summary of RNA-seq datasets obtained from different projects Note: * MA reads: reads mapped to more than one genomic locus

3.2 Identification of AS events

Mapping assembled mRNA transcripts, including ESTs, to the genome we identified a total of 2,098 AS events including 74 ExonS, 213 AltD, 397 AltA, 723 IntronR, and 691 complex events (Table 3). These AS events were generated from 1,804 genes involving 3,835 transcripts.

Table 3 Classification of alternative splicing events in different datasets in A. niger

For identification of AS events in RNA-seq data, we first identified AS events in each project by merging mapping information of all samples within each project, then we merged all mapping information of seven projects. Finally, we merged mRNA mapping information with RNA-seq data mapping information to generate the final list of AS events (Table 3). Since each project had different numbers of samples and associated reads, thus the AS events varied greatly among them. Among the basic AS events, AltA was the predominant AS type followed by IntronR type in all the RNA-seq projects (Table 3). However, we noticed that IntronR became predominant type when all RNA mapping data were merged. Another interesting observation was when RNA-seq data were combined with mRNA data, the total numbers of AS events were more than the addition of the two datasets analyzed individually, since AS events were identified by pair-wise comparisons of isoforms generated from a gene undergoing AS (Table 3). In short, in this work we have identified a total of 63,715 AS events including 10,097 (15.8%) AltA, 6,063 (9.5%) AltD, 12,469 (19.6%) IntronR, 1,945 (3.1%) ExonS, and 33,141 (52.0%) complex events. ExonS is the least basic AS type in A. niger, suggesting the splicing mechanism in fungal species is similar with plant species. These AS events were identified from 4,972 genomic loc involving 43,156 unique transcripts.

Combining all the mapping data we obtained a total of 9,939 genomic loci with a total of 66,007 transcripts assembled by Cufflinks tool (Table 4). Among these genomic loci, 7,026 (70.7%) loci produced two or more transcripts and 2,913 loci generated one transcript each locus. However, based on the loci mapping of the isoform transcripts with AS events, 4,972 loci were identified for generation of AS events. Thus, the AS rate based on current data collection at the genome level for all genes was estimated to be ~50.0% in A. niger (Supplementary Table 2). However, as there were 1,032 gens consisting of only a single exon, i. e., no intron, AS rate among intron containing genes was 55.8%. To our knowledge this is the highest AS rate ever reported in a fungal species (reviewed by Fang et al., 2020). Comparing with gene models in the reference genome annotation, 8,347 genomic loci in our work were mapped to the reference genomic loci. However, current A. niger reference genome was annotated with 10,828 genomic loci (Pel et al., 2007), interestingly, these loci were mapped to 8,347 genomic loci generated in our data. Clearly some of reference genomic loci were merged into longer, and, thus resulting in, fewer loci in our data. In addition, there were 1,592 genomic loci unmapped with annotated genomic loci, representing newly identified genomic loci with the supporting evidence from RNA-seq data. The newly identified genomic loci generated 3,388 transcripts and 2,795 ORFs were predicted from these transcripts.

3.3 Functional annotation of transcripts

A total of 66,007 RNA transcript sequences were retrieved and further annotated, including ORF prediction, functional annotation based on BLASTX against UniProt-Swiss-Prot database, and protein family (Pfam) prediction. These basic features of these transcripts were summarized (Table 4). The transcripts have an average length of 3,735 bp and 22,162 (33.6%) transcripts had a BLASTX match against Swiss-Prot data. A total of 61,153 (92.6%) were predicted to have an ORF region which contained a start codon with a minimum length of 20 amino acids encoded. The average length of predicted proteins was 285 amino acids. The current reference protein dataset in NCBI consists of 14,086 sequences with an average length 440 amino acids.  In addition, using BLASTN no-gap search with a cut-off of ≥97% identity and a minimum length of align 60 bp 27,756 (41.8%) transcripts were matched with transcripts of gene models in A. niger.

Table 4 Basic features of assembled RNA transcripts and functional annotation in A. niger

The predicted proteins from retrieved mRNA transcripts were annotated to Pfam, that facilitates examination if the functional domain in proteins encoded by different transcript isoforms is maintained. Since the isoforms in alternatively spliced genes may encode a truncated protein, thus resulting in a domain loss, due to a pre-mature stop codon or may not be able to translate to a protein due to a translation frame shift. A total of 19,177 predicted ORFs had a Pfam match (Table 4). Among 9,939 total genomic loci identified in this work, 2,941 loci encoded proteins had Pfam matched, and among them 2,000 genomic loci were alternatively spliced. We compared the Pfam of protein sequences encoded by the isoforms in these genomic loci subject to AS with at least one isoform having Pfam. Within these loci there were 13,914 transcripts encoded ORFs had Pfam match, however, among them 4,867 transcripts encoded ORFs with different Pfam in the same loci, and 4,485 transcripts encoded ORFs lost the Pfam, i. e. a functional domain (Supplementary Table 3). However, the impacts of AS events on the functionalities of different protein isoforms need to be validated experimentally.

To compare the AS rates of genes encoding different Pfam we extracted only one Pfam annotation for genes having multiple isoforms. Among a total of 9,939 genes (genomic loci), 2941 of them encoded at least one ORF matching to protein families. Among them 2,000 (68.0%) genes were alternatively spliced, though different gene families had variable AS rates (Table 5; Supplementary Table 4). The observed much higher AS rates in these protein coding genes having Pfam matches, particularly carbohydrate-active enzymes (CAZymes), indicate AS playing important roles in regulation of various types of cellular processes. For example, based on the CAZy classification we identified 84 genes encoding different families of CAZymes and found 52 of them were alternatively spliced (Table 6) (http://www.cazy.org/Home.html) (Drula et al., 2022). The functions of isoforms need to be further investigated in regards of carbohydrate metabolism in the fermentation process such as for biofuel production (Borin et al, 2017; Daly et al. 2017).

Table 5 Genomic loci encoding major protein families have variable alternatives splicing rates * Note: * Partial list. The full list can be found in supplementary table 4

Table 6 Majority of genes of CAZymes related to biomass degradation undergo alternative splicing in A. niger

3.4 Dynamic changes of AS events in response to different growth conditions

Gene expression is dynamically regulated by the compositional changes in the growth media. Daly et al. (2017), Borin et al. (2017) and van Munster et al. (2020) reported the gene expression changes of CAZymes, sugar transporters, and transcription factors and other proteins related to lignocellulose degradation in response to different growth substrates including wheat straw, feedstock Miscanthus, and sugarcane bagasse, respectively. Here we use data collected by Daly et al. (2017) and van Munster et al. (2020) to demonstrate the dynamic changes of AS events in gene expression. The treatments of growth substrates included glucose-rich conditions (GLU, control), hydrothermal pretreated Miscanthus (HTM), hydrothermal pretreated wheat straw (HTS), ionic liquid pretreated Miscanthus (ILM), ionic liquid pretreated straw (ILS), knife-milled Miscanthus (KMM), and knife-milled wheat straw (KMS) (Daly et al. 2017; van Munster et al. 2020). We combined all samples collected over a 5-day time course for each treatment for identification AS events, the results were summarized (Table 7). The differences in total numbers of AS events were mostly caused by the data size differences in these treatments (R² = 0.9603) (Figure 1). The similar trend of positive relationship between numbers of AS events and mapped RNA-seq reads was reported in our previous analysis with data collected from potato plants (Lee and Min, 2023). To detect the effects of different treatments on AS events, we carried out pairwise comparisons of AS events in these treatments of combined data (Figure 2, Figure 3, Figure 4). Clearly, AS events were dynamically changed with different treatment of growth substrates in glucose, differently pretreated Miscanthus, and differently pretreated wheat straws. There were conserved AS events among all treatments as well as treatments specifically generated AS events (Figure 2, Figure 3, Figure 4). Daly et al. (2017) and van Munster et al. (2020) reported dynamic transcriptomic expressions of CAZymes at gene levels in different pretreated growth substrates over a time course. Our analysis demonstrated AS events were also dynamically regulated in these treatments. More detailed analysis would allow to identify treatment specific AS events and novel isoforms for further exploration of novel enzymatic activities for applications in the biofuel production.

Table 7 Identification of alternative splicing events in different growth conditions in A. niger

Figure 1 Relationships of total mapped reads and total alternative splicing events identified in seven different treatments of growth substrates in A. niger

Figure 2 Alternative splicing events identified in glucose (GLU), hydrothermal pretreated Miscanthus (HTM), ionic liquid pretreated Miscanthus (ILM), and knife-milled Miscanthus (KMM) as substrates for A. niger culture

Figure 3 Alternative splicing events identified in glucose (GLU), hydrothermal pretreated wheat straw (HTS), ionic liquid pretreated straw (ILS), and knife-milled wheat straw (KMS) as substrates for A. niger culture

Figure 4 Alternative splicing events identified in glucose (GLU), wheat straw, and Miscanthus as substrates for A. niger culture

4 Discussion

We integrated mapping information of more than 10 billion of RNA-seq reads generated from 303 samples collected from eight recently published articles with 19,571 assembled transcripts of mRNA mapping information for identification of genome-wide AS events in A. niger. To our knowledge, this is the first large scale genome-wide meta-analysis of AS events in A. niger using RNA-seq data collected from various growth substrates. Combining all the mapping data generated a total of 9,939 genomic loci with a total of 66,007 transcripts assembled. A total of 63,715 AS events were identified from 4,972 genomic loc involving 43,156 unique transcripts. The AS rate based on current work was estimated to be about 50.0% in A. niger. We expect that more AS events and a higher AS rate can be obtained when more RNA-seq or transcripts data are integrated for genome mapping in A. niger in future. The impact of AS events on the encoded protein functions including enzymes needs to be evaluated individually. Our data including the assembled transcript sequences and mapping files are publicly available for the community to experimentally verify the identified isoform sequences and explore their functional novelties of enzymes for bioprocessing applications.

The work represents a large scale of genome-wide systematic identification of alternatively spliced genes and isoforms in A. niger. As in our analysis showed AS in genes in fungal species may be a common process, we recommend that researchers working in fungal species consider AS analysis when performing transcriptomic studies. However, it should be noted that these isoform transcript sequences were assembled by Cufflinks, validation by RT-PCR or cloning the full-length of mRNA transcripts are needed for further detailed functional analysis. The work carried out by Xu et al. (2024) in lignocellulos-degrading enzyme genes and enzyme variants in A. niger can serve as an example for this type of analysis. Computational identification of genes undergoing AS and annotation of their associated transcript isoform sequences are useful for researchers to design more specific experiments to examine the functions of genes of interests. The current work provides an important resource for investigating alternatively spliced genes and their associated functions of protein isoforms in A. niger. The data are expected to be useful in identifying homologous alternatively spliced genes in other fungal species in future research.

Author Contributions

XM designed the experiments. XM and FY provided the methodology, software support, and data analysis. XM, CJ, BL, MC, BW, CD, AP, PM, and KA carried out RNA-seq and mRNA transcript data collection and genome mapping. XM and FY prepared the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The Ohio Supercomputer Center provided computational resources for part of data processing.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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