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Results for “Protein Splicing”

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At least 19 recordsLinked to original sources

PubMed2026

Prenatal BPA exposure perturbs RNA-binding protein-mediated splicing regulation and synaptogenesis in the developing cerebellum in a sex-dependent manner.

BACKGROUND: Autism spectrum disorder (ASD) is a pervasive neurodevelopmental condition characterized by social communication deficits, exhibiting a male bias in prevalence. Emerging evidence suggests that prenatal exposure to bisphenol A (BPA) may perturb neurodevelopmental trajectories relevant to ASD. While the cerebellum is increasingly recognized as a brain region implicated in ASD pathophysiology, the impact of gestational BPA exposure on its post-transcriptional alternative splicing machinery remains fundamentally undefined. METHODS: Here, we investigated sex-dependent effects of prenatal BPA exposure on the alternative splicing landscape of the neonatal rat cerebellum. We utilized RNA-seq to profile differential alternative splicing (DAS) events. Ingenuity Pathway Analysis (IPA) was used to predict biological functions and canonical pathways, and to construct the interactome network of DAS genes. To explore candidate upstream regulatory mechanisms, we performed in silico molecular docking and used high-resolution melting (HRM) qRT-PCR to validate selected splicing events. Furthermore, we assessed in vitro cellular phenotypes in primary cerebellar neurons by measuring MTS-based viability and Syn1/Psd95 puncta colocalization. RESULTS: Prenatal BPA exposure was associated with widespread DAS in genes enriched for ASD-relevant pathways in the neonatal rat cerebellum. To our knowledge, this study is the first to report molecular docking analyses predicting favorable interactions between BPA and several candidate RNA-binding proteins (RBPs), including CPEB1, RALYL, HNRNPDL, and ACO1. Our findings support a model in which BPA may perturb RBP-associated splicing regulation, including altered splicing of chromatin regulators such as Ccar1 in males. These molecular and cellular findings were accompanied by sex-stratified differences in neuronal viability and synaptic puncta measurements. BPA exposure was associated with an increased MTS viability signal in male primary cerebellar neurons, together with significant reductions in Psd95 and Syn1 puncta density, whereas female neurons showed significantly increased synaptic puncta colocalization together with reduced viability. CONCLUSIONS: In this study, we propose that prenatal BPA may be relevant to ASD-related neurodevelopmental pathways through sex-dependent changes in RBP-associated alternative splicing, including altered splicing of Ccar1 in males, together with distinct cellular outcomes. Together, these findings identify the developing cerebellum as a sensitive target of prenatal BPA exposure and highlight alternative splicing as a candidate pathway relevant to ASD biology.

Animals↗

PubMed2026

Identification of a Functional CYP2C8 Variant Allele that Alters Splicing, Reduces Protein Expression, and Increases Drug Exposure.

This study investigated genetic determinants of the pharmacokinetics of the CYP2C8 index drugs repaglinide and gemfibrozil, and their interaction in healthy participants. Sequencing data from a study with montelukast revealed a novel functional CYP2C8 allele (rs2071426, CYP2C8*19), predicted to create an intronic splice donor site. In human liver samples, CYP2C8*19 associated with transcript-specific changes in CYP2C8 mRNA expression, reduced CYP2C8 protein expression, and decreased enzyme activity. Consistently, participants with the CYP2C8*19/*19 genotype had 45% greater area under the plasma repaglinide concentration-time curve from time zero to infinity (AUC0-∞) than participants with CYP2C8*1/*1 (P = 1.6 × 10-4). Participants with CYP2C8*1/*3 had 26% smaller AUC0-∞ (P = 0.0033) and those with CYP2C8*1/*4 had 51% greater AUC0-∞ (P = 8.2 × 10-4). The fold increase in repaglinide AUC0-∞ caused by gemfibrozil was 36% (P = 1.3 × 10-4) smaller in CYP2C8*19/*19 participants than in CYP2C8*1/*1 participants. In a genome-wide association study (GWAS), SLCO1B1 c.521 T>C (rs4149056) associated with increased repaglinide AUC0-∞ (P = 4.5 × 10-15; n = 172) and SLCO1A2 variants associated with decreased AUC0-∞ (P < 10-8). In a GWAS of repaglinide after gemfibrozil pretreatment, SLCO1C1 variants associated with decreased AUC0-∞ (P < 1.6 × 10-8; n = 66). Participants with the poor function SLCO1B1 genotype showed a 32% smaller fold increase in repaglinide AUC0-∞ following gemfibrozil than participants with the normal function SLCO1B1 genotype (P = 0.0045). This study characterizes CYP2C8*19 as a novel decreased function allele and shows that CYP2C8 and SLCO1B1 genotypes affect the gemfibrozil-repaglinide interaction.

Humans↗

PubMed2026

Cellular modifiers of TDP-43 phase transition and cytoplasmic aggregation.

RNA-binding protein TAR DNA-binding protein 43 (TDP-43) can form liquid-like nuclear assemblies whose phase behavior is thought to influence its aggregation propensity and neurotoxic activity. The mechanisms that govern the liquid-to-solid phase transition of TDP-43 remain poorly defined. Here, we combined chemical and genome-wide genetic screens to identify cellular factors that modulate the phase behavior of an RNA-binding-defective TDP-43 mutant. Our screens uncovered multiple cellular processes, including RNA splicing, protein translation, proteostasis imbalance, and nuclear export as TDP-43 phase regulators. We also developed a semi-permeabilized cell system that partially recapitulates the TDP-43 phase transition in vitro, and showed that nuclear export inhibition reshapes the nuclear environment to favor RNA-dependent liquid-liquid phase separation (LLPS) of TDP-43, which mitigates its aggregation. Nuclear export inhibition in a brain organoid model bearing an ALS-associated mutation reduces pathogenic phospho-TDP-43 accumulation. These findings identify multiple modulators of TDP-43 phase transitions in a sensitized model system and establish a framework for further dissecting the link between nuclear transport and TDP-43 phase dynamics.

DNA-Binding Proteins↗

PubMed2026

Nuclear export modulates TDP-43 phase transition and cytoplasmic aggregation.

RNA-binding protein TAR DNA-binding protein 43 (TDP-43) can form liquid-like, nuclear assemblies whose phase behavior may influence its aggregation propensity and neurotoxic activity. The mechanism(s) that modulates the transition of TDP-43 from a liquid to solid phase is poorly defined. Here we combine chemical and genome-wide genetic screenings to identify cellular factors that modulate the phase behavior of an RNA-binding defective TDP-43 mutant that mimics an Amyotrophic Lateral Sclerosis (ALS)-associated variant. Our screens uncover multiple cellular processes including RNA splicing, protein translation, proteostasis imbalance and nuclear export as TDP-43 phase regulators. Importantly, TDP-43 phase transition can be dynamically recapitulated in vitro in a semi-permeabilized cell system, which reveals that the inhibition of nuclear export reshapes the nuclear environment in favor of an RNA-dependent TDP-43 liquid-liquid phase separation (LLPS) state, which mitigates cytoplasmic TDP-43 aggregation. We validated this mechanism in a brain organoid model bearing an ALS-associated mutation, showing that nuclear export deficiency can limit pathogenic phospho-TDP-43 accumulation. These findings establish nuclear export as a key regulator of TDP-43 phase transitions and define a mechanistic framework that links altered nuclear transport and phase dynamics to TDP-43 aggregation potential.

Amyotrophic Lateral Sclerosis (ALS)↗

PubMed2024

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Animals↗

PubMed2026

Analysis of structure and conservation for supporting functional evaluation of PMS2 missense variants.

Germline defects in mismatch repair (MMR) genes are known to significantly increase the risk of developing certain types of cancers, notably colorectal and endometrial cancers. These conditions are characterized under Lynch syndrome. Accurate diagnosis of this predisposition, along with meaningful predictive testing for family members, necessitates the identification of pathogenic variants. However, classifying small coding genetic variants identified in cancer patients is very challenging, specifically in the case of PMS2 variants, since PMS2 pathogenic variants display a lower penetrance and less severe phenotype and therefore a lower tumor burden in affected families. We have assembled clinical data on four PMS2 missense variants of uncertain significance (VUS) identified in 23 patients (p.(Asp286Gly), p.(Asn335Ser), p.(Ile679Thr) and p.(Arg799Trp)). For these variants, functional testing was performed (RNA splicing, protein stability and catalytic activity). Since many protein ortholog sequences and accurate predictive models from AlphaFold2 are available, we also included a systematic analysis of residue conservation and structural role (ConStruct assessment). Overall, our findings indicate that p.(Asp286Gly) and p.(Arg799Trp) behave similarly to wild-type PMS2 and are thus probably neutral. In contrast, p.(Asn335Ser) and p.(Ile679Thr) conferred defects in protein expression or MMR activity. These could be explained by the relevant roles of these amino acids in MLH1-PMS2-N-terminal dimerization (p.Asn335) and C-terminal dimerization (p.Ile679). Our data thus suggest that p.(Asp286Gly) and p.(Arg799Trp) are benign, while the tumor risk in the other two variants remains to be established. Taken together, we suggest roadmaps for the individualized evaluation of difficult uncertain variants by comprising information from all available sources.

Humans↗

PubMed2026

[Genetic and functional characterization of a novel KIT splicing variant in a Chinese three-generation pedigree with piebaldism].

OBJECTIVES: To investigate the genetic etiology of a three-generation pedigree affected with piebaldism. METHODS: Next-generation sequencing and Sanger sequencing were employed to detect and verify gene variants. Bioinformatics tools were used to predict the effects of candidate variants on splicing and protein function. RT-PCR and Sanger sequencing were further performed to validate the impact of the variant on RNA splicing, and homology modeling was applied to predict its effect on the three-dimensional structure of the KIT protein. The pathogenicity of the variant was then classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the UK Association for Clinical Genomic Science (ACGS). RESULTS: A heterozygous insertion variant near the splice site, c.1990+8_1990+9insTGCACCATTGGAGGTAAA, was identified in the KIT gene in the proband and was found to co-segregate with the phenotype within the family. RT-PCR and cDNA sequencing revealed that this variant led to aberrant splicing during transcription, resulting in a 21 bp in-frame insertion in the mRNA, which encodes an extra 7 amino acids within the tyrosine kinase domain and may thus affect protein function. In silico predictions, together with the experimental findings, supported classification of this variant as likely pathogenic according to relevant variant interpretation guidelines. CONCLUSIONS: The heterozygous splice-site insertion variant KIT:c.1990+8_1990+9insTGCACCATTGGAGGTAAA is the genetic cause of piebaldism in this pedigree.

Genetics diagnosis↗

PubMed2025

iSoMAs: Finding isoform expression and somatic mutation associations in human cancers.

Aberrant alternative splicing, prevalent in cancer, impacts various cancer hallmarks involving proliferation, angiogenesis, and invasion. Splicing disruption often results from somatic point mutations rewiring functional pathways to support cancer cell survival. We introduce iSoMAs (iSoform expression and somatic Mutation Association), an efficient computational pipeline leveraging principal component analysis technique, to explore how somatic mutations influence transcriptome-wide gene expression at the isoform level. Applying iSoMAs to 33 cancer types comprising 9,738 tumor samples in The Cancer Genome Atlas, we identified 908 somatically mutated genes significantly associated with altered isoform expression across three or more cancer types. Mutations linked to differential isoform expression occurred through both cis- and trans-acting mechanisms, involving well-known oncogenes/suppressor genes, RNA binding protein and splicing factor genes. With wet-lab experiments, we verified direct association between TP53 mutations and differential isoform expression in cell cycle genes. Additional iSoMAs genes have been validated in the literature with independent cohorts and/or methods. Despite the complexity of cancer, iSoMAs attains computational efficiency via dimension reduction strategy and reveals critical associations between regulatory factors and transcriptional landscapes.

Humans↗

PubMed2026

Regulation of TET function by PROSER1 in development and hematologic malignancies.

Ten eleven translocation (TET) proteins are central regulators of DNA methylation homeostasis and play essential roles in development and disease, including hematopoietic malignancies. Among the three TET family members, mutations in TET2 are frequently observed in hematologic disorders. TET enzymes catalyze the iterative oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, enabling DNA demethylation. Beyond catalysis, TET proteins also perform important non-enzymatic functions mediated through interactions with diverse protein partners, highlighting the importance of defining their regulatory interactome. Previous studies identified several TET-associated factors, including O-Linked N-acetylglucosamine transferase (OGT), members of the Drosophila behavior/human splicing (DBHS) protein family, and proline and serine-rich protein 1 (PROSER1). However, these interactions were largely considered independently. Recent findings now demonstrate that TET proteins, OGT, PROSER1, and DBHS proteins assemble into a higher-order regulatory unit termed the TOPD (TET-OGT-PROSER1-DBHS) complex. In this review, we discuss how TOPD provides a conceptual framework for understanding multicomponent regulation of TET function, spatial control of DNA demethylation, and maintenance of epigenetic homeostasis, with implications for developmental syndromes and hematopoiesis.

Humans↗

PubMed2025

Balancing LncRNA H19 and miR-675 Bioconversion as a Key Regulator of Embryonic Myogenesis Under Maternal Obesity.

BACKGROUND: Maternal obesity (MO) impairs fetal skeletal muscle development, but the underlying mechanisms remain poorly defined. The regulatory roles of lncRNA H19 and its first exon derived microRNA675 (miR675) in prenatal muscle development remain to be examined. H19/Igf2 are in the same imprinting cluster with H19 expressed from the maternal allele while Igf2 expresses paternally. H19 contains a G-rich loop, and KH-type splicing regulatory protein (KHSRP) mediates the biogenesis of pre-miRNAs containing G-rich loops, which depends on its phosphorylation by AKT, a key mediator of IGF2 signalling. This study aims to depict the elusive function of these regulators that are affected by MO during embryonic myogenesis. METHODS: Single-cell transcriptomic sequencing and GeoMx spatial RNA sequencing were performed to identify the differentially expressed genes between embryos from MO and control (CT) mice. Both E11.5 and E13.5 embryos were collected and analysed to validate the sequencing data. The roles of H19 and miR657 in myogenesis were further analysed in P19 embryonic cells via CRISPR/dCas9-mediated H19 activation and inhibition. The epigenetic changes of H19 were analysed by methylated DNA immunoprecipitation, and allele-targeted analysis of H19 was performed by crossing C57BL/6J and CAST/EiJ mice. RESULTS: Transcriptomic analysis showed that MO embryos contained less differentiated myocytes (1.34%) than CT embryos (2.86%). Myogenesis-related GO biological processes were down-regulated in the MO embryonic myotome region. MO embryos showed lower expression of myogenic transcription factors such as Myf5, Myod1, Myog, Mef2c and Myh3 (p < 0.05). MO altered epigenetic modifications of the H19 genomic cluster, showing a decreased methylation level in H19 imprinting control region (p < 0.05) and a diallelic expression pattern of H19, which elevated its expression in MO embryos. Overexpression of H19 inhibited myogenesis in P19 cells, but miR675 promoted myogenesis, suggesting the critical regulatory roles of bioconversion of H19 to miR675. A KHSRP mediates the biogenesis of miR675, a process that relies on its phosphorylation by IGF2/AKT signalling. Knocking-down of KHSRP and inhibition of AKT abolished miR675 biogenesis. MO suppressed IGF2/AKT signalling and blocked KHSRP-dependent miR675 biogenesis in embryos. CONCLUSIONS: We found differential effects of H19 and miR675 on embryonic myogenesis. MO up-regulates H19 but blocks its miR675 bioconversion via suppressing IGF2/AKT/KHSRP signalling axis. Myogenesis in MO embryos was impeded due to the highly accumulated H19 and blocked miR675 biogenesis.

RNA, Long Noncoding↗

PubMed2026

Meta-PseU: A meta-classifier for robust prediction of RNA pseudouridine modification sites from long sequences.

BACKGROUND AND OBJECTIVES: Pseudouridine (Ψ) represents one of the most abundant and conserved RNA modifications. Ψ provides an additional hydrogen-bond donor that enhances RNA structural stability and modulates translation. It participates in diverse biological processes, including RNA-protein interactions, splicing, translational control, and stress responses. Aberrant pseudouridylation is implicated in cancer, neurodegenerative disorders, and autoimmune diseases. Despite its biological importance, experimental identification of Ψ sites remains time-consuming and costly, limiting the feasibility of transcriptome-wide profiling. Computational approaches have therefore become essential complements to experimental techniques. However, state-of-the-art machine-learning and deep-learning predictors often suffer from limited generalizability due to small training datasets. To overcome these issues, we aim at constructing new long-sequence datasets and developing a novel Ψ site predictor. METHODS: New long-sequence datasets were constructed as benchmarks for RNA Ψ-site prediction. The Ψ modification sites in RMBase 3.0 were mapped to the reference genomes across three species of human, mouse, and yeast, and the RNA sequences with a length of 201 were generated by extending the upstream and downstream from the mapped, central sites. To eliminate sequence redundancy, the sequences were clustered using CD-HIT with a 70% sequence identity threshold. We developed Meta-PseU, a logistic regression-based meta-classifier that considered 118 machine learning and deep learning classifiers. The datasets and programs are freely accessible at https://github.com/kuratahiroyuki/MetaPseU. RESULTS: By optimizing model configuration, we proposed the Meta-PseU model stacking 32 machine learning and deep learning classifiers out of 118 classifiers. Meta-PseU substantially improved model generalizability, overcoming a key limitation of existing approaches. It greatly outperformed state-of-the-art predictors and achieved increasing accuracy with increasing sequence length. CONCLUSIONS: Long-sequence datasets were newly constructed as benchmarks for RNA Ψ-site prediction. Meta-PseU offers a new framework for robust Ψ-site identification by using long sequences.

Pseudouridine↗

PubMed2026

The Germline SH2B3rs111340708 Splicing Variant Drives Intron Retention and Protein Instability by Impacting Clinical Outcomes in Core Binding Factor AML.

The SH2B3 gene, also known as LNK, encodes an adaptor protein that negatively regulates key hematopoietic signaling pathways, including JAK-STAT, MAPK, and PI3K/AKT, thereby maintaining hematopoietic homeostasis. SH2B3 interacts with major signaling regulators such as JAK2, MPL, FLT3, and KIT. Loss-of-function alterations have been reported in several hematologic malignancies, supporting its role as a leukemia predisposition gene. We previously identified a germline start-loss mutation (c.3G > A) in SH2B3 in a family with early-onset myeloproliferative neoplasm, demonstrating that this variant causes SH2B3 haploinsufficiency. In the present study, next-generation sequencing of 149 de novo AML patients identified a frequent intronic polymorphism (rs111340708), located within intron 6 (IVS6) of SH2B3. Although this variant has a reported minor allele frequency (MAF) of approximately 12% in European populations, it was enriched in our AML cohort, reaching 34.2% in Core Binding Factor leukemias (CBFLs). The presence of the rs111340708 variant was associated with inferior overall survival, whereas no significant association with progression-free survival was observed. Functional analyses demonstrated that this polymorphism promotes aberrant IVS6 intron retention in AML cells, resulting in reduced abundance of correctly spliced SH2B3 transcripts and predicted generation of truncated peptides and/or nonsense-mediated decay. Consistently, immunoblot analyses of AML patient samples and hematologic cell lines revealed heterogeneous SH2B3 protein expression, including additional SH2B3-immunoreactive species in variant carriers, together with reduced levels of the canonical SH2B3 protein. Collectively, these findings identify a common germline splicing polymorphism as a novel mechanism contributing to SH2B3 functional impairment in AML and highlight the potential relevance of non-coding variants in leukemia pathogenesis, with possible implications for risk stratification and future therapeutic strategies.

Humans↗

PubMed2026

The Role of Small Segmental Duplications in Generating Identical Isoforms Through Alternative Splicing Sites.

Alternative splicing plays a crucial role in expanding proteomic diversity but can also generate identical isoforms under certain conditions. While mutually exclusive splicing of tandem exons has occasionally been reported to produce identical isoforms, the extent to which other splicing events contribute to this phenomenon remains unclear. In this study, we demonstrate that alternative 5' and 3' splice site selection can also lead to the formation of identical isoforms, providing an additional type of splicing event for functional redundancy in transcriptomes. To address this, we analyzed reference genome annotations from 15 plant species, including Arabidopsis thaliana and wheat (Triticum aestivum), obtained from the RefSeq database. Identical isoforms were computationally defined as transcripts with distinct exon-intron structures but identical coding sequences. Our analysis reveals that the majority of alternative 5' and 3' fragments originate from small segmental duplications, suggesting that sequence repetition within gene regions facilitates the emergence of such splicing patterns. We also observed differences in the annotated 5' UTRs of some identical isoforms. However, since the alternative splicing sites themselves were not located within UTRs, these differences may reflect annotation uncertainty rather than genuine AS-derived variation. Given that UTR predictions in reference databases are not always precise, such observations should be interpreted cautiously. Expression analysis using an isoform-specific k-mer approach confirmed that identical isoforms can be differentially regulated. These findings suggest that, beyond expanding protein diversity, alternative splicing can also generate redundant isoforms that are differentially expressed at the RNA level, indicating potential regulatory roles. By elucidating the structural and regulatory factors contributing to the formation and retention of identical isoforms, our study provides new insights into the evolutionary and functional significance of alternative splicing in plants.

Alternative Splicing↗

PubMed2025

The proxiome of a plant viral protein with dual targeting to mitochondria and chloroplasts revealed MAPK cascade and splicing components as proviral factors.

The coat protein (CP) of the melon necrotic spot virus (MNSV) is a multifunctional factor localized in the chloroplast, mitochondria, and cytoplasm, playing a critical role in overcoming plant defenses such as RNA silencing (RNAi) and the necrotic hypersensitive response. However, the molecular mechanisms through which CP interferes with plant defenses remain unclear. Identifying viral-host interactors can reveal how viruses exploit fundamental cellular processes and help elucidate viral survival strategies. Here, we employed a TurboID-based proximity labeling approach to identify interactors of both the wild-type MNSV CP and a cytoplasmic CP mutant lacking the dual transit peptide (ΔNtCP). Of the interactors, eight were selected for silencing. Notably, silencing MAP4K SIK1 and NbMAP3Kε1 kinases, and a splicing factor homolog NbSMU2 significantly reduced MNSV accumulation, suggesting a proviral role for these proteins in plants. Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed the CP and ΔNtCP interaction with NbSMU2 and NbMAP3Kε1 but not with NbSIK1, which interacted with NbMAP3Kε1. These findings open up new possibilities for exploring how MNSV CP might modulate gene expression and MAPK, thereby facilitating MNSV infection.

Chloroplasts↗

PubMed2025

The modern expansion of Dscam1 isoform diversity in Drosophila is linked to fitness and immunity.

Drosophila melanogaster Down Syndrome cell adhesion molecule 1 (Dscam1) gene encodes 38,016 diverse cell surface receptor proteins via alternative splicing, which have both nervous and immune functions. However, it remains elusive why organisms have evolved such an astonishing diversity of isoforms. Here, we show that fitness and immunity properties have driven the modern evolution of Dscam1 isoform diversity. We assess multiple aspects of fly fitness in deletion mutants harboring exon 4, 6, or 9 clusters, respectively, reducing ectodomain isoform diversity stepwise from 18,612 to 396. All fitness-related traits generally improved as the potential number of isoforms increased; however, the magnitude of the changes varied remarkably in a variable cluster-specific manner. Correlation analysis revealed that fitness-related traits were much more sensitive to reductions in Dscam1 diversity compared to canonical neuronal self/non-self discrimination. We conclude that the role of Dscam1 isoforms in canonical neuronal self-avoidance and self/non-self discrimination is mediated by a small fraction of all isoforms (<1/10), whereas a separate role essential for other developmental contexts and resistances, likely in fitness and immunity, requires almost full isoform diversity. Thus, fitness and immunity properties, rather than canonical neuronal functions, are the dominant drivers during the modern diversification of the Dscam1 isoform. Our findings suggest that Dscam1 diversity is closely linked to adaptation and species diversification in arthropods.

Animals↗

PubMed2025

Proteomics-based evaluation of AAV dystrophin gene therapy outcomes in mdx skeletal muscle.

Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease characterized by loss of dystrophin protein. Therapeutic attempts to restore a functional copy of dystrophin to striated muscle are under active development, and many utilize adeno-associated viral (AAV) vectors. However, the limited cargo capacity of AAVs precludes delivery of full-length dystrophin, a 427 kDa protein, to target tissues. Recently, we developed a method to express large dystrophin constructs using the protein trans-splicing mechanism mediated by split inteins and myotropic AAV vectors. The efficacy of this approach to restore muscle function in mdx4cv mice was previously assessed using histology, dystrophin immunolabeling, and Western blotting. Here, we expand our molecular characterization of dystrophin constructs with variable lengths using a mass spectrometry-based proteomics approach, providing insight into unique protein expression profiles in skeletal muscles of wild-type, dystrophic mdx4cv, and AAV-treated mdx4cv mice. Our data reveal several affected cellular processes in mdx4cv skeletal muscles with changes in the expression profiles of key proteins to muscle homeostasis, whereas successful expression of dystrophin constructs results in an intermediate to complete restoration. This study highlights several biomarkers that could be used in future preclinical or clinical studies to evaluate the effectiveness of therapeutic strategies.

Animals↗

PubMed2025

Alternative Splicing in Mechanically Stretched Podocytes as a Model of Glomerular Hypertension.

KEY POINTS: Mechanical stretch induced over 3000 alternative splicing events in podocytes, affecting gene expression and protein abundance. Seventeen genes showed consistent splicing events across multiple analysis tools, with key isoform changes. Shroom3 and Myl6 underwent isoform switches under mechanical stretch, altering the C-terminal sequence and interaction properties of Myl6. BACKGROUND: Alterations in pre-mRNA splicing are crucial to the pathophysiology of various diseases. However, the effects of alternative splicing of mRNA on podocytes in hypertensive nephropathy are still unknown. The Sys_CARE project aimed to identify alternative splicing events involved in the development and progression of glomerular hypertension. METHODS: Murine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA sequencing, and several bioinformatic alternative splicing tools. RESULTS: Using transcriptomic and proteomic analysis, we identified significant changes in gene expression and protein abundance because of mechanical stretch. RNA-Seq identified over 3000 alternative spliced genes after mechanical stretch, including all types of alternative splicing events. Among these, 17 genes exhibited an alternative splicing event across four different splicing analysis tools. From this group, we focused on Myl6, a component of the myosin protein complex, and Shroom3, an actin-binding protein essential for podocyte function. We identified two Shroom3 isoforms with significant expression changes under mechanical stretch, which was validated by quantitative RT-PCR and in situ hybridization. In addition, we observed an expression switch of two Myl6 isoforms after mechanical stretch, accompanied by an alteration in the C-terminal amino acid sequence. CONCLUSIONS: A comprehensive RNA-Seq analysis of mechanically stretched podocytes identified novel potential podocyte-specific biomarkers and highlighted significant alternative splicing events, notably in the mRNA of Shroom3 and Myl6.

podocyte↗

PubMed2026

Proteogenomic features define subtypes of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a biologically heterogeneous B-cell malignancy. Although genomics and transcriptomics have delineated parts of the MCL disease spectrum, proteomics remains largely unexplored. Here, we conducted a comprehensive proteogenomic analysis integrating genomics, transcriptomics, and proteomics on peripheral blood samples from 27 patients with MCL and 4 healthy donors to investigate the translational and posttranslational dimensions of MCL. Our study identified 1296 downregulated and 468 upregulated proteins in MCL cells. The splicing pathways were significantly upregulated at both the mRNA and protein levels, suggesting a critical role for aberrant RNA splicing in MCL pathogenesis. Integration of proteomic data with genetic aberrations revealed immunoglobulin heavy chain variable mutational status and CCND1 mutation are associated with distinctive transcriptomic and proteomic profiles, which correspond to significant differences in clinical outcomes. A multiomics molecular stratification model incorporating proteomic data showed superior predictive power for patient survival compared with single-omics models (concordance index, 0.83 vs 0.74). This study provides, to our knowledge, the first comprehensive proteogenomic profile of MCL, offering novel insights into its molecular mechanisms and clinical behavior. The identification of molecular subtypes and prognostic protein signatures underscores the potential of proteomics to guide precision medicine strategies for MCL.

Humans↗