Intron Retention Coupled with Nonsense-Mediated Decay is Involved in Cellulase Biosynthesis in Cellulolytic Fungi

Abstract Background Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in ehancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. Result We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to non cellulase-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. Conclusion All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

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A Dynamic Intron Retention Program in the Mammalian Megakaryocyte and Erythrocyte Lineages

Blood ◽

10.1182/blood.v126.23.2380.2380 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2380-2380

Author(s):

Christopher R Edwards ◽

Rob Middleton ◽

Xiuli An ◽

Tejaswini Mishra ◽

Narla Mohandas ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Conflicts Of Interest ◽

Intron Retention ◽

Expression Patterns ◽

Erythroid Differentiation ◽

Cell Types ◽

Mrna Levels ◽

Protein Coding ◽

Nonsense Mediated Decay

Abstract Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing. Disclosures No relevant conflicts of interest to declare.

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Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

Applied and Environmental Microbiology ◽

10.1128/aem.00653-20 ◽

2020 ◽

Vol 86 (15) ◽

Author(s):

Dan Liu ◽

Yisong Liu ◽

Duoduo Zhang ◽

Xiaoting Chen ◽

Qian Liu ◽

...

Keyword(s):

Neurospora Crassa ◽

Filamentous Fungi ◽

Ribosome Biogenesis ◽

Strain Improvement ◽

Cellulase Production ◽

Protein Processing ◽

Mrna Levels ◽

Industrial Enzymes ◽

Content Type ◽

Proteome Profiling

ABSTRACT Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases. IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

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Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi

mBio ◽

10.1128/mbio.00277-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 8

Author(s):

Lara Hassan ◽

Liangcai Lin ◽

Hagit Sorek ◽

Laura E. Sperl ◽

Thomas Goudoulas ◽

...

Keyword(s):

Filamentous Fungi ◽

Regulatory Networks ◽

Plant Cell Wall ◽

Competitive Inhibition ◽

Plant Biomass ◽

Degradation Products ◽

Cellulase Production ◽

Content Type ◽

Industrial Strains ◽

Genomics Tools

ABSTRACTIt is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available forNeurospora crassato investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCEIn fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

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Comparative transcriptome analysis of Trichoderma reesei reveals different gene regulatory networks induced by synthetic mixtures of glucose and β-disaccharide

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00411-4 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Yonghao Li ◽

Jingze Yu ◽

Peng Zhang ◽

Tingting Long ◽

Yi Mo ◽

...

Keyword(s):

Transcription Factors ◽

Trichoderma Reesei ◽

Regulatory Networks ◽

Expression Profiles ◽

Low Cost ◽

Strain Improvement ◽

Gene Expression Profiles ◽

Cellulase Production ◽

Mitogen Activated Protein Kinase ◽

Lignocellulose Degradation

AbstractThe mixture of glucose and β-disaccharide (MGD) synthesized by transglycosylation of glucose as a low-cost soluble carbon source can efficiently induce cellulase production in Trichoderma reesei, which holds potential for the biorefining of lignocellulosic biomass. However, it is not yet fully understood how MGD induces T. reesei cellulase. In this study, transcriptomic analyses were conducted to investigate the molecular basis of MGD for lignocellulose-degrading enzyme production of T. reesei Rut C30 compared with that on lactose. Particular attention was paid to CAZymes, transcription factors, transporters and other protein processing pathways related to lignocellulose degradation. As a result, MGD can elicit transcription of GH5-, GH6- and GH7-encoding cellulases that is up to 1.4-fold higher than that induced by lactose, but GH11- and GH74-encoding xylanases are downregulated by 1.7- and 4.4-fold, respectively. Gene expression profiles suggest that the transcription activators xyr1 and vib1 are significantly upregulated and that the mitogen-activated protein kinase pathway is strengthened compared to the case of lactose induction. In addition, hac1-encoding UPR-specific transcription factors are significantly upregulated by MGD, which may be enhanced due to proper folding and processing of nascent proteins. These findings provide a theoretical basis for further understanding the characterization of efficient cellulase production using MGD as an inducer in T. reesei and offer potential strategies for strain improvement.

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Regulation of plant gene expression by alternative splicing

Biochemical Society Transactions ◽

10.1042/bst0380667 ◽

2010 ◽

Vol 38 (2) ◽

pp. 667-671 ◽

Cited By ~ 24

Author(s):

Craig G. Simpson ◽

Sujatha Manthri ◽

Katarzyna Dorota Raczynska ◽

Maria Kalyna ◽

Dominika Lewandowska ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Protein Function ◽

Cell Types ◽

Splicing Factors ◽

Mrna Levels ◽

Sr Protein ◽

Nonsense Mediated Decay ◽

Cis Acting ◽

Reverse Transcription Pcr

AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed ‘combinatorial’ control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)–PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT–PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process.

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spliceR: An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data.

10.7287/peerj.preprints.80v1 ◽

2013 ◽

Author(s):

Kristoffer Vitting-Seerup ◽

Bo T Porse ◽

Albin Sandelin ◽

Johannes E Waage

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Stop Codon ◽

Intron Retention ◽

Digital Gene Expression ◽

Exon Skipping ◽

R Package ◽

Nonsense Mediated Decay ◽

Coding Potential

With the advent of increasing depth and decreasing costs in digital gene expression technologies exemplified by RNA-sequencing, researchers are now able to profile the transcriptome with unprecedented detail. These advances not only allow for precise approximation of gene expression levels, but also for characterization of alternative isoform usage/switching between samples. Recent software improvements in full transcript deconvolution prompted us to develop spliceR , an R package for classification of alternative splicing. spliceR labels isoforms based on fully assembled transcripts, detecting single- and multiple exon skipping, alternative donor or acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. Alongside, event spliced-in/out values are calculated for effective post-filtering, and genomic coordinates of differentially spliced elements are annotated for downstream sequence analysis. Furthermore, spliceR has the option to predict the coding potential and thereby the nonsense mediated decay (NMD) sensitivity of transcripts based on stop codon position.

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mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway

Nucleic Acids Research ◽

10.1093/nar/gkz500 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6900-6916 ◽

Cited By ~ 8

Author(s):

Xue-hai Liang ◽

Joshua G Nichols ◽

Chih-Wei Hsu ◽

Timothy A Vickers ◽

Stanley T Crooke

Keyword(s):

Gene Expression ◽

Antisense Oligonucleotides ◽

Mrna Levels ◽

Independent Manner ◽

Nonsense Mediated Decay ◽

Coding Regions ◽

Antisense Approach ◽

Modulation Of Gene Expression ◽

Reduce Gene Expression ◽

Increase Gene Expression

Abstract Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2′-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3′ end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.

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Cross-talk of cellulose and mannan perception pathways leads to inhibition of cellulase production in several filamentous fungi

10.1101/520130 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lara Hassan ◽

Liangcai Lin ◽

Hagit Sorek ◽

Thomas Goudoulas ◽

Natalie Germann ◽

...

Keyword(s):

Filamentous Fungi ◽

Cross Talk ◽

Regulatory Networks ◽

Plant Cell Wall ◽

Competitive Inhibition ◽

Plant Biomass ◽

Degradation Products ◽

Cellulase Production ◽

Cellulase Expression ◽

Genomics Tools

AbstractIt is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual signaling pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of “confusion” due to cross-talk between the perception pathways for cellulose and the hemicellulose mannan in several filamentous fungi, leading to the inhibition of cellulase expression. We used the functional genomics tools available forNeurospora crassato investigate this signaling overlap at the molecular level. Cross-talk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.

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