In multicellular organisms, a pre-mRNA is composed of exons intervened with introns. The interspersed introns can be removed through a large multiprotein splicing complex (the spliceosome) to yield the mature mRNA. During this splicing processing, a pre-mRNA can generate one or more functional transcripts by the alternative splicing (AS) process . AS is a well-conserved mechanism for producing multiple isoforms of mRNA and/or proteins to increase the diversity of the transcriptome and proteome under varied physiological circumstances, therefore overcoming the limitations caused by a finite genome and aiding an organism in accommodating to the changing environment . There are seven main types of AS events: mutually exclusive, intron retention, cassette exons, alternative initiation, alternative termination, alternative donors, and alternative acceptors .
In intron retention (IR), introns that are supposed to be spliced appear in mature mRNA and subsequently participate in the translation process. At first, IR had been considered useless resulting from the malfunctioning of the spliceosome, and has been relatively ignored. Nevertheless, the role of IR has been studied more extensively in recent years. Retained introns affect the localization, translatability, stability, and function of the transcripts containing them . IR plays an essential role in the regulations of gene expression, mRNA localization, tissue-specific protein diversity, alternative splicing, and dosage compensation of the X chromosome, participating in biological events such as stress response, development, tissue differentiation, and disease [5, 6]. An increasing research has demonstrated that intron retention is widely found in animals, plants and fungi, serving as one of the effective strategies for post transcriptional regulation in eukaryotes . In fungi, IR is reported to be involved in fungal cell complexity, pathogenicity , heat shock response , the nutrient sensing such as glucose , and nitrogen source . However, the function of IR in fungi has not been well studied, as compared to the extensive study of IR in animals and plants.
The intron-containing transcripts usually accommodate one or more premature termination codons (PTCs), which allow them to be recognized and degraded by nonsense-mediated mRNA decay (NMD), inhibiting the production of potentially harmful proteins . The coupling of IR with NMD (IR-NMD) poses an additional post-transcriptional regulatory layer that can control mRNA quality and gene expression level . IR-NMD can regulate a gene function via upregulating the expression of a non-functional NMD-targeted isoform of the gene, and consequently reducing the translation of the protein . IR-NMD is rarely studied in filamentous fungi.
Cellulose is found widely in nature (leaves, grass, and wood) and waste materials (municipal wastes and agricultural wastes). The cellulase-mediated bioconversion of cellulose to fermentable sugars for biomass-derived biorefinery is potent, sustainable, and environment-friendly. Cellulase is a mixture of extracellular enzymes acting collaboratively for cellulose decomposition, majorly including endoglucanase (CMC; EC 184.108.40.206) cleaving cellulose in an endo-acting way and exhibiting a great affinity towards the soluble cellulose derivatives, cellobiohydrolase (CBH; EC 220.127.116.11) working as exoenzymes to generate cellobiose from cellulose, and β-glucosidase (BGL; EC 18.104.22.168) converting cellobiose to glucose [13, 14]. Cellulose is the efficient natural inducer for cellulase production by filamentous fungi like Trichoderma reesei, Neurospora crassa, Aspergillus nidulans, and Penicillium decumbens, followed by lactose, while glucose is the repressor of cellulase production. The knowledge on the regulatory molecular mechanism of cellulase synthesis in filamentous fungi is prerequisite for rationally engineering fungal strains to improve the production of cellulase and other proteins like pharmaceutical proteins and industrial enzymes, which has received increasing attention . It has been reported that cellulase production is regulated by varied signal pathways, like carbon catabolite repression (CCR) , calcium signal transduction pathway , and the TOR pathway . When glucose exists, CCR is induced through the transcription factor CRE1 to almost completely block the cellulase production . In contrast, the Ca2+ burst through calcium signal transduction pathway can strengthen the cellulase production and cell metabolism . However, whether IR and NMD is involved in cellulase biosynthesis in filamentous fungi and how, remain totally unknown.
In this study, to investigate the function of IR-NMD in cellulase production in filamentous fungi, the mRNA levels and IR rates of the three major cellulase genes (cel7a, cel7b, and cel3a) in T. reesei, an well-known working horse for industrial cellulase production , were investigated under cellulase-(non)producing conditions together with the mRNA levels of the NMD pathway. A lower IR level with a more active NMD pathway was observed under cellulase-producing than under cellulase-nonproducing, suggesting a key role of IR-NMD in cellulase biosynthesis. To further verify this finding, the NMD pathway inhibitor was explored to repress the NMD pathway, leading to the increased IR rates and decreased mRNA levels of cellulase genes as well as significantly decreased cellulase production on cellulose. Moreover, the effect of the NMD pathway inhibitor on the phenotype of T. reesei and the TOR pathway was investigated. This study provides new knowledge on the regulation mechanism of cellulase production in terms of IR-NMD.