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Alternative splicing (AS) is a key post-transcriptional modification that helps in increasing protein diversity. Almost 90% of the protein-coding genes in humans are known to undergo AS and code for different transcripts. Some transcripts are associated with diseases such as breast cancer, lung cancer and glioblastoma. Hence, these transcripts can serve as novel therapeutic and prognostic targets for drug discovery. Herein, we have developed a pipeline, Finding Alternative Splicing Events (FASE), as the R package that includes modules to determine the structure and concentration of transcripts using differential AS. To predict the correct structure of expressed transcripts in given conditions, FASE combines the AS events with the information of exons, introns and junctions using graph theory. The estimated concentration of predicted transcripts is reported as the relative expression in terms of log2CPM. Using FASE, we were able to identify several unique transcripts of EMILIN1 and SLK genes in the TCGA-BRCA data, which were validated using RT-PCR. The experimental study demonstrated consistent results, which signify the high accuracy and precision of the developed methods. In conclusion, the developed pipeline, FASE, can efficiently predict novel transcripts that are missed in general transcript-level differential expression analysis. It can be applied selectively from a single gene to simple or complex genome even in multiple experimental conditions for the identification of differential AS-based biomarkers, prognostic targets and novel therapeutics., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show that ceramide synthase 2 (CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.

Alternative splicing (AS)-a process by which a single gene gives rise to different protein isoforms in eukaryotes-has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans , has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3 , which contains a single intron. The sod3 Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans IMPORTANCE The emergence of resistance in Candida albicans , an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans , here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress., (Copyright © 2020 Muzafar et al.)

Alternative splicing (AS) coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here we show that, during dietary restriction (DR), AS is enhanced in Caenorhabditis elegans and mice. A splicing mediator hrpu-1 regulates a significant part of these AS events in C. elegans; knocking it down suppresses DR-mediated longevity. Concurrently, due to increased AS, NMD pathway genes are upregulated and knocking down UPF1 homologue smg-2 suppresses DR lifespan. Knockdown of NMD during DR significantly increases the inclusion of PTC-containing introns and the lengths of the 3'UTRs. Finally, we demonstrate that PHA-4/FOXA transcriptionally regulates the AS-NMD genes. Our study suggests that DR uses AS to amplify the proteome, supporting physiological remodelling required for enhanced longevity. This increases the dependence on NMD, but also helps fine-tune the expression of metabolic and splicing mediators. AS-NMD may thus provide an energetically favourable level of dynamic gene expression control during dietary restriction.Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here, the authors provide evidence that AS-NMD is enhanced during dietary restriction (DR) and is required for DR-mediated longevity assurance in C. elegans.

The splicing factors RDM16 and STA1 have been reported to play a role in the RNA directed DNA Methylation (RdDM) pathway. In this pathway, small interfering RNAs guide de-novo methylation of homologous DNA sequences. DNA methylation is epigenetic marks, which can suppress transposable elements, repeat sequences and genes. It also affects the chromatin structure. Upon deletion of RDM16 and STA1 , previous studies based on gene level analysis were unable to find differentially spliced events implicated in RdDM pathway. In this study, RNA-seq data from RDM16 & STA1 mutants were analyzed. Differential expression analysis was performed at exon and intron level. This analysis revealed 3474 genes with potential differential expression events in the RDM16 mutant and 3058 in the STA1 mutant. We found 17 genes that have been reported to be involved in the RdDM pathway. These results suggest involvement of RDM16 and STA1 by influencing the expression of key gene components such as MORC6 in the RdDM pathway. Therefore, this study increases the understanding of the role of RDM16 and STA1 splicing factors in DNA methylation. [ABSTRACT FROM AUTHOR]