Mapping alternative splicing events in colorectal cancer.

Although aberrant splicing events of genes are closely related to the development and progression of colorectal cancer (CRC), the mapping of abnormal splicing events, especially alternative splicing (AS) event types and the underlying effects, remain investigational. In the present study, we analyzed a public RNA-seq database (GSE138202) and identified 14,314 significant AS events in CRC patients compared to healthy individuals. Most of the key genes such as oncogenes involved in the development of CRC have different AS event types. Moreover, the results demonstrate that certain AS events may play a significant role in the functioning of key genes involved in splicing factors and microRNAs. Furthermore, we observed that the oncogene CDK4 in CRC tends to undergo exon 2 skipping AS events, resulting in a stronger tendency for protein expression to form complexes with CCND1, thereby inhibiting the cell cycle and weakening cell proliferation, while enhancing cell migration capability. These findings not only provide new insights into the mechanism of AS in regulating CRC, but also offers a theoretical basis for targeted splicing therapy in CRC.

RBM7 deficiency promotes breast cancer metastasis by coordinating MFGE8 splicing switch and NF-kB pathway.

Aberrant alternative splicing is well-known to be closely associated with tumorigenesis of various cancers. However, the intricate mechanisms underlying breast cancer metastasis driven by deregulated splicing events remain largely unexplored. Here, we unveiled that RBM7 is decreased in lymph node and distant organ metastases of breast cancer as compared to primary lesions and low expression of RBM7 is correlated with the reduced disease-free survival of breast cancer patients. Breast cancer cells with RBM7 depletion exhibited an increased potential for lung metastasis compared to scramble control cells. The absence of RBM7 stimulated breast cancer cell migration, invasion, and angiogenesis. Mechanistically, RBM7 controlled the splicing switch of MFGE8, favoring the production of the predominant isoform of MFGE8, MFGE8-L. This resulted in the attenuation of STAT1 phosphorylation and alterations in cell adhesion molecules. MFGE8-L exerted an inhibitory effect on the migratory and invasive capability of breast cancer cells, while the truncated isoform MFGE8-S, which lack the second F5/8 type C domain had the opposite effect. In addition, RBM7 negatively regulates the NF-κB cascade and an NF-κB inhibitor could obstruct the increase in HUVEC tube formation caused by RBM7 silencing. Clinically, we noticed a positive correlation between RBM7 expression and MFGE8 exon7 inclusion in breast cancer tissues, providing new mechanistic insights for molecular-targeted therapy in combating breast cancer.

Identification of Alternatively Spliced Novel Isoforms of Human HSPB8 Gene.

HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics.

Effects of Gene Alternative Splicing Events on Resistance to Cryptocaryonosis of Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker (L. crocea) is a productive species in marine aquaculture with great economic value in China. However, the sustainable development of large yellow croaker is hampered by various diseases including cryptocaryonosis caused by Cryptocaryon irritans. The genetic regulation processes for cryptocaryonosis in large yellow croaker are still unclear. In this present study, we analyzed differential alternative splicing events between a C. irritans resistance strain (RS) and a commercial strain (CS). We identified 678 differential alternative splicing (DAS) events from 453 genes in RS and 719 DAS events from 500 genes in CS. A set of genes that are specifically alternatively spliced in RS was identified including mfap5, emp1, and trim33. Further pathway analysis revealed that the specifically alternative spliced genes in RS were involved in innate immune responses through the PRR pathway and the Toll and Imd pathway, suggesting their important roles in the genetic regulation processes for cryptocaryonosis in large yellow croaker. This study would be helpful for the studies of the pathogenesis of cryptocaryonosis and dissection of C. irritans resistance for L. crocea.

Synonymous variant at the terminal nucleotide in exon 3 of F7 causes abnormal splicing: A case report.

BACKGROUND: Synonymous variants are non-pathogenic due to non-substitution of amino acids. However, synonymous exonic terminal nucleotide substitutions may affect splicing. Splicing variants are easily analyzed at RNA level for genes expressed in blood cells. Minigene analysis provides another method for splicing variant analysis of genes that are poorly or not expressed in peripheral blood. METHODS: Whole exome sequencing was performed to screen for potential pathogenic mutations in the proband, which were validated within the family by Sanger sequencing. The pathogenicity of the synonymous mutation was analyzed using the minigene technology. RESULTS: The proband harbored the compound heterogeneous variants c. [291G >A; 572-50C >T] and c.681 + 1G >T in F7, of which the synonymous variant c.291G >A was located at the terminal position of exon 3. Minigene analysis revealed exon3 skipping due to this mutation, which may have subsequently affected protein sequence, structure, and function. CONCLUSION: Our finding confirmed the pathogenicity of c.291G >A, thus extending the pathogenic mutation spectrum of F7, and providing insights for effective reproductive counseling.

Post-transcriptional regulation of DEAD-box RNA helicases in hematopoietic malignancies.

Hematopoiesis represents a meticulously regulated and dynamic biological process. Genetic aberrations affecting blood cells, induced by various factors, frequently give rise to hematological tumors. These instances are often accompanied by a multitude of abnormal post-transcriptional regulatory events, including RNA alternative splicing, RNA localization, RNA degradation, and storage. Notably, post-transcriptional regulation plays a pivotal role in preserving hematopoietic homeostasis. The DEAD-Box RNA helicase genes emerge as crucial post-transcriptional regulatory factors, intricately involved in sustaining normal hematopoiesis through diverse mechanisms such as RNA alternative splicing, RNA modification, and ribosome assembly. This review consolidates the existing knowledge on the role of DEAD-box RNA helicases in regulating normal hematopoiesis and underscores the pathogenicity of mutant DEAD-Box RNA helicases in malignant hematopoiesis. Emphasis is placed on elucidating both the positive and negative contributions of DEAD-box RNA helicases within the hematopoietic system.

Splicing Machinery Is Impaired in Oral Squamous Cell Carcinomas and Linked to Key Pathophysiological Features.

Alternative splicing dysregulation is an emerging cancer hallmark, potentially serving as a source of novel diagnostic, prognostic, or therapeutic tools. Inhibitors of the activity of the splicing machinery can exert antitumoral effects in cancer cells. We aimed to characterize the splicing machinery (SM) components in oral squamous cell carcinoma (OSCC) and to evaluate the direct impact of the inhibition of SM-activity on OSCC-cells. The expression of 59 SM-components was assessed using a prospective case-control study of tumor and healthy samples from 37 OSCC patients, and the relationship with clinical and histopathological features was assessed. The direct effect of pladienolide-B (SM-inhibitor) on the proliferation rate of primary OSCC cell cultures was evaluated. A significant dysregulation in several SM components was found in OSCC vs. adjacent-healthy tissues [i.e., 12 out of 59 (20%)], and their expression was associated with clinical and histopathological features of less aggressiveness and overall survival. Pladienolide-B treatment significantly decreased OSCC-cell proliferation. Our data reveal a significantly altered expression of several SM-components and link it to pathophysiological features, reinforcing a potential clinical and pathophysiological relevance of the SM dysregulation in OSCC. The inhibition of SM-activity might be a therapeutic avenue in OSCC, offering a clinically relevant opportunity to be explored.

Genetic variation mining of the Chinese mitten crab (Eriocheir sinensis) based on transcriptome data from public databases.

At present, public databases house an extensive repository of transcriptome data, with the volume continuing to grow at an accelerated pace. Utilizing these data effectively is a shared interest within the scientific community. In this study, we introduced a novel strategy that harnesses SNPs and InDels identified from transcriptome data, combined with sample metadata from databases, to effectively screen for molecular markers correlated with traits. We utilized 228 transcriptome datasets of Eriocheir sinensis from the NCBI database and employed the Genome Analysis Toolkit software to identify 96 388 SNPs and 20 645 InDels. Employing the genome-wide association study analysis, in conjunction with the gender information from databases, we identified 3456 sex-biased SNPs and 639 sex-biased InDels. The KOG and KEGG annotations of the sex-biased SNPs and InDels revealed that these genes were primarily involved in the metabolic processes of E. sinensis. Combined with SnpEff annotation and PCR experimental validation, a highly sex-biased SNP located in the Kelch domain containing 4 (Klhdc4) gene, CHR67-6415071, was found to alter the splicing sites of Klhdc4, generating two splice variants, Klhdc4_a and Klhdc4_b. Additionally, Klhdc4 exhibited robust expression across the ovaries, testes, and accessory glands. The sex-biased SNPs and InDels identified in this study are conducive to the development of unisexual cultivation methods for E. sinensis, and the alternative splicing event caused by the sex-biased SNP in Klhdc4 may serve as a potential mechanism for sex regulation in E. sinensis. The analysis strategy employed in this study represents a new direction for the rational exploitation and utilization of transcriptome data in public databases.

Identification of pancreatic adenocarcinoma immune subtype associated with tumor neoantigen from aberrant alternative splicing.

Background: Pancreatic adenocarcinoma (PAAD) is referred to as an immunologically "cold" tumor that responds poorly to immunotherapy. A fundamental theory that explains the low immunogenicity of PAAD is the dramatically low tumor mutation burden (TMB) of PAAD tumors, which fails to induce sufficient immune response. Alternative splicing of pre-mRNA, which could alter the proteomic diversity of many cancers, has been reported to be involved in neoantigen production. Therefore, we aim to identify novel PAAD antigens and immune subtypes through systematic bioinformatics research. Methods: Data for splicing analysis were downloaded from The Cancer Genome Atlas (TCGA) SpliceSeq database. Among the available algorithms, we chose CIBERSORT to evaluate the immune cell distribution among PAADs. The TCGA-PAAD expression matrix was used to construct a co-expression network. Single-cell analysis was performed based on the Seurat workflow. Results: Integrated analysis of aberrantly upregulated genes, alternatively spliced genes, genes associated with nonsense-mediated RNA decay (NMD) factors, antigen presentation and overall survival (OS) in TCGA-PAAD revealed that PLEC is a promising neoantigen for PAAD-targeted therapy. We identified a C2 TCGA-PAAD subtype that had better prognosis and more CD8+ T-cell infiltration. We propose a novel immune subtyping system for PAAD to indicate patient prognosis and opportunities for immunotherapy, such as immune checkpoint (ICP) inhibitors. Conclusions: In conclusion, the present study used a transcriptome-guided approach to screen neoantigen candidates based on alternative splicing, NMD factors, and antigen-presenting signatures for PAAD. A prognosis model with guidance of immunotherapy will aid in patient selection for appropriate treatment.

Rbm24 modulates neuronal RNA splicing to restrict cognitive dysfunction.

Synaptic dysfunction is associated with early neurodegenerative changes and cognitive deficits. Neuronal cell-specific alternative splicing (AS) programs exclusively encode unique neuron- and synapse-specific proteins. However, it remains unclear whether splicing disturbances in neurons influence the pathogenesis of cognitive impairment. Here, we observed that RNA-binding motif protein 24 (RBM24) expression was decreased in Alzheimer's disease (AD) patients. Using conditional RBM24 knockout mice, we demonstrated that deletion of RBM24 in the brain resulted in learning and memory impairment. Electrophysiological recordings from hippocampal slices from mice lacking RBM24 revealed multiple defects in excitatory synaptic function and plasticity. Furthermore, RNA sequencing and splicing analysis showed that RBM24 regulates a network of genes related to cognitive function. Deletion of RBM24 disrupted the AS of synapse-associated genes, including GluR2 and Prrt1, the major disease genes involved in cognitive impairment and memory loss, leading to cognitive dysfunction. Together, our results suggest that the regulation of mRNA splicing by RBM24 is a key process involved in maintaining normal synaptic function and provide novel mechanistic insights into the pathogenesis of AD.

Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model - A multi-platform analysis using mass spectrometry and RT-qPCR.

Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78-91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling via integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in in vitro models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and in vitro models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.

CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Disentangling the splicing factor programs underlying complex molecular phenotypes.

The regulation of exon inclusion through alternative splicing tunes the cell's behavior by increasing the functional diversity of the transcriptome and the proteome. Splicing factors work in concert to generate gene isoform pools that contribute to cell phenotypes yet their activity is controlled by multiple regulatory and signaling layers. This hinders identification of functional, phenotype-specific splicing factors using traditional single-omic measurements, such as their mutational state or expression. To address this challenge, we propose repurposing the virtual inference of protein activity by enriched regulon analysis (VIPER) to measure splicing factor activity solely from their downstream exon transcriptomic inclusion signatures. This approach is effective in assessing the effect of co-occurring splicing factor perturbations, as well as their post-translational regulation. As proof of concept, we dissect recurrent splicing factor programs underlying tumorigenesis including aberrantly activated factors acting as oncogenes and inactivated ones acting as tumor suppressors, which are undetectable by more conventional methodologies. Activation and inactivation of these cancer splicing programs effectively stratifies overall survival, as well as cancer hallmarks such as proliferation and immune evasion. Altogether, repurposing network-based inference of protein activity for splicing factor networks distills common, functionally relevant splicing programs in otherwise heterogeneous molecular contexts.

SimSpliceEvol2: alternative splicing-aware simulation of biological sequence evolution and transcript phylogenies.

BACKGROUND: SimSpliceEvol is a tool for simulating the evolution of eukaryotic gene sequences that integrates exon-intron structure evolution as well as the evolution of the sets of transcripts produced from genes. It takes a guide gene tree as input and generates a gene sequence with its transcripts for each node of the tree, from the root to the leaves. However, the sets of transcripts simulated at different nodes of the guide gene tree lack evolutionary connections. Consequently, SimSpliceEvol is not suitable for evaluating methods for transcript phylogeny inference or gene phylogeny inference that rely on transcript conservation. RESULTS: Here, we introduce SimSpliceEvol2, which, compared to the first version, incorporates an explicit model of transcript evolution for simulating alternative transcripts along the branches of a guide gene tree, as well as the transcript phylogenies inferred. We offer a comprehensive software with a graphical user interface and an updated version of the web server, ensuring easy and user-friendly access to the tool. CONCLUSION: SimSpliceEvol2 generates synthetic datasets that are useful for evaluating methods and tools for spliced RNA sequence analysis, such as spliced alignment methods, methods for identifying conserved transcripts, and transcript phylogeny reconstruction methods. The web server is accessible at https://simspliceevol.cobius.usherbrooke.ca , where you can also download the standalone software. Comprehensive documentation for the software is available at the same address. For developers interested in the source code, which requires the installation of all prerequisites to run, it is provided at  https://github.com/UdeS-CoBIUS/SimSpliceEvol .

DeepSplice: a deep learning approach for accurate prediction of alternative splicing events in the human genome.

Alternative splicing (AS) is a crucial process in genetic information processing that generates multiple mRNA molecules from a single gene, producing diverse proteins. Accurate prediction of AS events is essential for understanding various physiological aspects, including disease progression and prognosis. Machine learning (ML) techniques have been widely employed in bioinformatics to address this challenge. However, existing models have limitations in capturing AS events in the presence of mutations and achieving high prediction performance. To overcome these limitations, this research presents deep splicing code (DSC), a deep learning (DL)-based model for AS prediction. The proposed model aims to improve predictive ability by investigating state-of-the-art techniques in AS and developing a DL model specifically designed to predict AS events accurately. The performance of the DSC model is evaluated against existing techniques, revealing its potential to enhance the understanding and predictive power of DL algorithms in AS. It outperforms other models by achieving an average AUC score of 92%. The significance of this research lies in its contribution to identifying functional implications and potential therapeutic targets associated with AS, with applications in genomics, bioinformatics, and biomedical research. The findings of this study have the potential to advance the field and pave the way for more precise and reliable predictions of AS events, ultimately leading to a deeper understanding of genetic information processing and its impact on human physiology and disease.

Identification of antennal alternative splicing by combining genome and full-length transcriptome analysis in Bactrocera dorsalis.

Alternative splicing is an essential post-transcriptional regulatory mechanism that diversifies gene function by generating multiple protein isoforms from a single gene and act as a crucial role in insect environmental adaptation. Olfaction, a key sense for insect adaptation, relies heavily on the antennae, which are the primary olfactory organs expressing most of the olfactory genes. Despite the extensive annotation of olfactory genes within insect antennal tissues facilitated by high-throughput sequencing technology advancements, systematic analyses of alternative splicing are still relatively less. In this study, we focused on the oriental fruit fly (Bactrocera dorsalis), a significant pest of fruit crops. We performed a detailed analysis of alternative splicing in its antennae by utilizing the full-length transcriptome of its antennal tissue and the insect's genome. The results revealed 8600 non-redundant full-length transcripts identified in the oriental fruit fly antennal full-length transcriptome, spanning 4,145 gene loci. Over 40% of these loci exhibited multiple isoforms. Among these, 161 genes showed sex-biased isoform switching, involving seven different types of alternative splicing. Notably, events involving alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS) were the most common. Of all the genes undergoing ATSS and ATTS alternative splicing between male and female, 32 genes were alternatively spliced in protein coding regions, potentially affecting protein function. These genes were categorized based on the length of the sex-biased isoforms, with the highest difference in isoform fraction (dIF) associated with the ATSS type, including genes such as BdorABCA13, BdorCAT2, and BdorTSN3. Additionally, transcription factor binding sites for doublesex were identified upstream of both BdorABCA13 and BdorCAT2. Besides being expressed in the antennal tissues, BdorABCA13 and BdorCAT2 are also expressed in the mouthparts, legs, and genitalia of both female and male adults, suggesting their functional diversity. This study reveals alternative splicing events in the antennae of Bactrophora dorsalis from two aspects: odorant receptor genes and other types of genes expressed in the antennae. This study not only provides a research foundation for understanding the regulation of gene function by alternative splicing in the oriental fruit fly but also offers new insights for utilizing olfaction-based behavioral manipulation techniques to manage this pest.

Rare variations within the serine/arginine-rich splicing factor PtoRSZ21 modulate stomatal size to determine drought tolerance in Populus.

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus.

An N-terminal and ankyrin repeat domain interactome of Shank3 identifies the protein complex with the splicing regulator Nono in mice.

An autism-associated gene Shank3 encodes multiple splicing isoforms, Shank3a-f. We have recently reported that Shank3a/b-knockout mice were more susceptible to kainic acid-induced seizures than wild-type mice at 4 weeks of age. Little is known, however, about how the N-terminal and ankyrin repeat domains (NT-Ank) of Shank3a/b regulate multiple molecular signals in the developing brain. To explore the functional roles of Shank3a/b, we performed a mass spectrometry-based proteomic search for proteins interacting with GFP-tagged NT-Ank. In this study, NT-Ank was predicted to form a variety of complexes with a total of 348 proteins, in which RNA-binding (n = 102), spliceosome (n = 22), and ribosome-associated molecules (n = 9) were significantly enriched. Among them, an X-linked intellectual disability-associated protein, Nono, was identified as a NT-Ank-binding protein. Coimmunoprecipitation assays validated the interaction of Shank3 with Nono in the mouse brain. In agreement with these data, the thalamus of Shank3a/b-knockout mice aberrantly expressed splicing isoforms of autism-associated genes, Nrxn1 and Eif4G1, before and after seizures with kainic acid treatment. These data indicate that Shank3 interacts with multiple RNA-binding proteins in the postnatal brain, thereby regulating the homeostatic expression of splicing isoforms for autism-associated genes after birth.

A chromosome-level reference genome assembly and a full-length transcriptome assembly of the giant freshwater prawn (Macrobrachium rosenbergii).

The giant freshwater prawn (Macrobrachium rosenbergii) is a key species in the aquaculture industry in several Asian, African and South American countries. Despite a considerable growth in its production worldwide, the genetic complexities of M. rosenbergii various morphotypes pose challenges in cultivation. This study reports the first chromosome-scale reference genome and a high-quality full-length transcriptome assembly for M. rosenbergii. We employed the PacBio High Fidelity (HiFi) sequencing to obtain an initial draft assembly and further scaffolded it with the chromatin contact mapping (Hi-C) technique to achieve a final assembly of 3.73-Gb with an N50 scaffold length of 33.6 Mb. Repetitive elements constituted nearly 60% of the genome assembly, with simple sequence repeats and retrotransposons being the most abundant. The availability of both the chromosome-scale assembly and the full-length transcriptome assembly enabled us to thoroughly probe alternative splicing events in M. rosenbergii. Among the 2,041 events investigated, exon skipping represented the most prevalent class, followed by intron retention. Interestingly, specific isoforms were observed across multiple tissues. Additionally, within a single tissue type, transcripts could undergo alternative splicing, yielding multiple isoforms. We believe that the availability of a chromosome-level reference genome for M. rosenbergii along with its full-length transcriptome will be instrumental in advancing our understanding of the giant freshwater prawn biology and enhancing its molecular breeding programs, paving the way for the development of M. rosenbergii with valuable traits in commercial aquaculture.

RNA-seq analysis reveals transcriptome reprogramming and alternative splicing during early response to salt stress in tomato root.

Salt stress is one of the dominant abiotic stress conditions that cause severe damage to plant growth and, in turn, limiting crop productivity. It is therefore crucial to understand the molecular mechanism underlying plant root responses to high salinity as such knowledge will aid in efforts to develop salt-tolerant crops. Alternative splicing (AS) of precursor RNA is one of the important RNA processing steps that regulate gene expression and proteome diversity, and, consequently, many physiological and biochemical processes in plants, including responses to abiotic stresses like salt stress. In the current study, we utilized high-throughput RNA-sequencing to analyze the changes in the transcriptome and characterize AS landscape during the early response of tomato root to salt stress. Under salt stress conditions, 10,588 genes were found to be differentially expressed, including those involved in hormone signaling transduction, amino acid metabolism, and cell cycle regulation. More than 700 transcription factors (TFs), including members of the MYB, bHLH, and WRKY families, potentially regulated tomato root response to salt stress. AS events were found to be greatly enhanced under salt stress, where exon skipping was the most prevalent event. There were 3709 genes identified as differentially alternatively spliced (DAS), the most prominent of which were serine/threonine protein kinase, pentatricopeptide repeat (PPR)-containing protein, E3 ubiquitin-protein ligase. More than 100 DEGs were implicated in splicing and spliceosome assembly, which may regulate salt-responsive AS events in tomato roots. This study uncovers the stimulation of AS during tomato root response to salt stress and provides a valuable resource of salt-responsive genes for future studies to improve tomato salt tolerance.