Computational Molecular Biology 2025, Vol.15, No.1, 1-12 http://bioscipublisher.com/index.php/cmb 1 Research Article Open Access Genome-Wide Identification and Analysis of Alternative Splicing in Aspergillus niger Xiangjia Min 1 , Courtney Jones 1, Burrows Logan 1, Maria Campean 1, Bilal Wekhyan 1, Chetan Dasana 1, Aaryan Patel 1, Pasan Mudiyanselage 2, Kolade Adeyemo 1, FengYu2 1 Department of Chemical and Biological Sciences, Youngstown State University, OH 44555 2 Department of Computer Science, Information, and Engineering Technology, Youngstown State University, OH 44555 Corresponding author: xmin@ysu.edu Computational Molecular Biology, 2025, Vol.15, No.1 doi: 10.5376/cmb.2025.15.0001 Received: 08 Nov., 2024 Accepted: 23 Dec., 2024 Published: 15 Jan., 2025 Copyright © 2025 Min et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Min X.J., Jones C., Logan B., Campean M., Wekhyan B., Dasana C., Patel A., Mudiyanselage P., Adeyemo K., and Yu F., 2025, Genome-wide identification and analysis of alternative splicing in Aspergillus niger, Computational Molecular Biology, 15(1): 1-12 (doi: 10.5376/cmb.2025.15.0001) Abstract Aspergillus niger is a widely used fungal species in fermentation industry. Identifying genes having RNA transcripts undergoing alternative splicing (AS) is important for understanding the gene expression regulations and finding novel enzymes for bioprocessing applications. In this study, we combined genome mapping information of all available RNA sequences and expressed sequence tags in the public database with RNA-seq data collected from 303 publicly available samples for identification of AS events in A. niger. We identified a total of 63,715 AS events including 10,097 (15.8%) alternative acceptor sites (AltA), 6,063 (9.5%) alternative donor sites (AltD), 12,469 (19.6%) intron retention sites (IntronR), 1,945 (3.1%) exon skipping sites (ExonS), and 33,141 (52.0%) complex events which contained two or more basic events in pairs of compared isoform transcripts. These AS events were identified from 4,972 genes involving 43,156 unique transcripts. The AS rate among all expressed genes was estimated to be ~50.0% in A. niger. Protein coding genes having protein family matches were estimated having 68.0% AS rate, including 52 of 84 genes coding for carbohydrate degrading enzymes (CAZymes) alternatively spliced. The functions of these proteins encoded by alternatively splicing generated isoforms need to be further investigated. We also identified a total of 1,592 new genomic loci with 3,388 transcripts that were not annotated in the reference genome. The AS data and genomic mapping data collected in this study provide a resource for further exploration of novel genes and enzymes inA. niger. Keywords Aspergillus niger; Alternative splicing; mRNA; RNA-seq; Carbohydrate active enzymes 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
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