Search Results (3,846)

Background/objectives: Obtaining a genetic diagnosis for patients with inherited retinal diseases has become even more important since gene-specific therapies have become available. When genetic screening reveals variants of uncertain significance (VUS), additional evidence is required to determine genetic eligibility for therapy. Confirming the effect on splicing that is predicted by SpliceAI could change their classification to either likely pathogenic or pathogenic and would therefore be of great importance when interpreting these variants when geneticists worldwide are trying to reach a diagnosis. Methods: Using minigene assays, we established a pipeline to assess the effect on splicing for all variants. We selected 73 RPE65 variants that were classified as either VUS or likely benign in the RPE65 Leiden Open Variant Database (LOVD) or ClinVar and were predicted to affect splicing by SpliceAI with a delta score of >0.1 and by using an analysis window of 5000 bp up- and downstream of the variant. Results: Using four wild-type vectors, we generated 59 constructs containing the variants of interest. Through these minigene assays, we assessed the effect on splicing of these VUS to enable reclassification. Upon quantification, we identified seven variants with a full, aberrant splicing effect without residual wild-type transcript. Eleven variants had between 5% and 20% remaining wild-type transcript. Forty-one variants had ≥20% residual wild-type transcript, among which fifteen variants showed no effect on splicing. Conclusions: Following the 2023 established ClinGen specific ACMG guidelines for RPE65 (Criteria Specification Registry), evidence from splice assays enabled reclassification of seven RPE65 variants from VUS to pathogenic through an assigned PVS1-very-strong criterium, as less than 5% of wild-type transcript was present. These findings contribute to the interpretation of variants observed in patients, which will in turn dictate their eligibility for gene therapy. Full article

The AP2/ERF transcription factor family plays pivotal roles in plant growth, stress responses, and defense mechanisms, yet its diversity in camphor trees remains underexplored. This study identified 154 AP2/ERF genes in the Camphora officinarum genome, with over 80% belonging to the ERF subfamily, a distribution consistent with other angiosperms. Synteny analysis revealed that tandem and segmental duplications were key drivers of family expansion, suggesting adaptive diversification under ecological pressures. Structural analysis showed that the majority of ERF/RAV subfamily genes possess a single-exon structure, whereas AP2 subfamily genes display muti-exon structures, indicating divergent evolutionary trajectories and potential functional versatility via alternative splicing. Promoter analyses detected numerous hormone- and stress-responsive elements, linking these genes to abscisic acid, auxin, gibberellin signaling, and pathogen defense. Further expression profiling during stem development showed that approximately 60% of CoAP2/ERF genes were constitutively expressed across 17 expression trends, suggesting roles in basal development and stage-specific processes (e.g., lignification). Under Epicoccum poaceicola infection, 23 CoAP2/ERF genes were differentially expressed. Among them, upregulated ERF homologs related to RAP2.2/2.3 suggested roles in hypoxia and antimicrobial responses, while downregulation of ERF5 homologs indicated a growth–defense trade-off, whereby developmental processes are suppressed to prioritize pathogen resistance. Overall, this study deciphers the genomic architecture and structural diversity of CoAP2/ERF genes, along with expression dynamics of these genes in development and biotic stress adaptation of camphor trees. These findings provide critical insights into transcriptional regulation of development and stress responses in camphor trees and establish a theoretical basis for molecular breeding and biotechnological strategies aimed at improving stress resilience in woody plants. Full article

MicroRNAs (miRNAs) are well known for regulating translation in the cytoplasm, yet their nuclear roles remain poorly understood. Previously, we identified spliceosome-associated miRNAs implicated in tumorigenesis and metastasis in breast cancer models. Here, we investigate their nuclear functions in the immortalized human cortical neuron (HCN) cell line, along with glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cell lines, both widely used as models for brain cancer research. Our findings reveal that spliceosome-associated miRNAs mark neuronal cancer cells and uncover novel nuclear targets. Notably, some spliceosomal miRNAs exhibit opposing regulatory effects in the nucleus compared to the cytoplasm, while others demonstrate potential novel nuclear functions. A prominent example is miR-99b, which overlaps the 5′ splice junction of the poorly characterized long non-coding RNA (lncRNA)sperm acrosome-associated 6 antisense RNA1 (SPACA6-AS1) and, through base pairing, enhances SPACA6-AS1 pre-mRNA levels. These results highlight the diverse and context-dependent functions of nuclear miRNAs in gene regulation and cancer progression, broadening our understanding of their regulatory potential beyond the cytoplasm. Full article

Leptin, an adipocyte-derived hormone, plays a central role in the regulation of energy homeostasis by acting on distinct hypothalamic nuclei. This review explores recent advances in our understanding of leptin’s region-specific actions within the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and lateral hypothalamus, highlighting their contributions to appetite regulation, energy expenditure, and neuroendocrine function. In the hypothalamic arcuate nucleus, leptin’s differential regulation of pro-opiomelanocortin and agouti-related peptide/neuropeptide Y neurons is now complemented by the identification of novel leptin-responsive neuronal populations—such as those expressing prepronociceptin, basonuclin 2, and Pirt—as well as a growing array of cellular and molecular modulators, including secreted factors like angiopoietin-like growth factor, zinc-α2-glycoprotein, and spexin, intracellular regulators such as Rap1, growth factor receptor-bound protein 10, and spliced X-box binding protein 1. In the ventromedial hypothalamus, leptin integrates with both peripheral (e.g., cholecystokinin) and central (e.g., pituitary adenylate cyclase-activating polypeptide) signals, while epigenetic mechanisms, such as those mediated by Jumonji domain-containing protein D3, regulate leptin receptor expression and sensitivity. The dorsomedial hypothalamus is increasingly recognized for coordinating leptin’s effects on metabolism, circadian rhythms, and respiration through distinct neuronal populations, including a subset of neurons co-expressing GLP-1 receptors that mediate leptin’s metabolic effects. In the lateral hypothalamus, leptin modulates reward-driven feeding via GABAergic neuronal populations—circuits that are particularly susceptible to disruption following early life trauma. Together, these insights reveal a sophisticated neurobiological framework through which leptin orchestrates systemic physiology. Understanding the heterogeneity of leptin signaling opens new avenues for restoring leptin sensitivity and developing personalized therapeutic strategies to combat obesity and related metabolic disorders. Full article

Background: The outgrowth of castration-resistant prostate cancer (CRPC) dictates patient morbidity and mortality. Recurrence of prostate cancer (PC) following androgen-deprivation therapy (ADT) often occurs due to constitutively active androgen receptor (AR) splice variants (AR-Vs), primarily AR-V7. Therefore, safe and effective therapies enabling the suppression of both full-length AR (AR-FL) and AR-Vs are urgently needed. The natural compound dimethyl sulfoxide (DMSO) has negligible cytotoxicity at concentrations below 5% and has anticancer potential. DMSO has been broadly used in biomedical research as a solvent for pharmaceuticals, as a cryoprotectant for cells, and as a topical treatment to suppress pain and inflammation. We investigated the effect of low-dose DMSO on AR expression, cell viability, and metastatic ability in PC cell lines expressing both AR-FL and AR-V7 (e.g., 22Rv1) and those expressing only AR-FL (e.g., C4-2B). Methods: MTT cell viability assays were performed to measure DMSO-induced cytotoxicity. Wound-healing assays were conducted to monitor the effect of DMSO on the migratory phenotype of cancer cells. Western blot analyses were performed to study the efficacy of DMSO in suppressing the protein levels of AR-FL and AR-V7, and expression of heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) was measured as a possible mechanism. Results: At concentrations of 0.1–1% (v/v), DMSO treatment showed minimal cytotoxicity, whereas the highest concentration used (2.5%) showed approximately 20% cytotoxicity at 96 h. Interestingly, however, DMSO treatment at concentrations of 1.0 and 2.5% significantly inhibited the migration of PC cells. Treatment with DMSO led to a dose-dependent inhibition of both AR-FL and AR-V7. Notably, in 22Rv1 cells, DMSO potently downregulated the expression of hnRNPH1, a splicing factor often associated with AR expression and signaling. Conclusions: Our findings suggest that low concentrations of DMSO may have potential as an effective anticancer agent, both at the initial and later stages when PC cells become castration resistant. Full article

Splicing regulatory sequences are cornerstones for exon recognition. Mutations that modify them can severely compromise mRNA maturation and protein production. A wide range of mutations, including SNPs and InDels, can influence splicing regulatory signals either directly (e.g., altering canonical donor and acceptor dinucleotides) or indirectly (e.g., creating cryptic splice sites). CSN1S1 and CSN1S2 genes encode for the two main milk proteins, αs1 and αs2 caseins, respectively. They represent a remarkable and unique example of the possibilities for alternative splicing of individual genes, both due to the high number of alternative splices identified to date and for recognized allele-specific splicing events. To date, at least 13 alleles of CSN1S1 originating from mutations that affect canonical splice sites have been described in Bos taurus (CSN1S1 A, A1, and H), Ovis aries (E, H, and I), Capra hircus (D and G), Bubalus bubalis (E, F) and Camelidae (A, C, and D). Similarly, allele-specific splicing events have been described at the CSN1S2 locus in B. taurus. (CSN1S2 D), C. hircus (CSN1S2 D), B. bubalis (CSN1S2 B, B1, and B2), Equus asinus (CSN1S2 I B), and Camelidae. This review highlights that mutations affecting canonical splice sites, particularly donor sites, are significant sources of genetic variation impacting the casein production of the main dairy livestock species. Currently, a key limitation on this topic is the lack of detailed functional and proteomic studies. Future research should leverage advanced omics technologies like long-read transcriptomics and allele-resolved RNA sequencing to characterize these splicing mechanisms, guiding precision breeding strategies. Full article

Rett syndrome is a severe neurodevelopmental disorder that occurs primarily in females and is caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene located on the X chromosome. Though MECP2 acts as a representative transcriptional regulator and affects gene expression both directly and indirectly, a complete understanding of this disease and the treatment mechanism has not been established yet. MECP2 plays a particularly important role in synaptic development, neuronal maturation, and epigenetic regulation in the brain. In this study, we summarize the molecular structure of MECP2, mutation-specific pathogenesis, and the role of MECP2 in regulating chromatin remodeling, RNA splicing, and miRNA processing to provide a comprehensive understanding of Rett syndrome. Additionally, we describe abnormal phenotypes manifested in various brain regions and other tissues owing to MECP2 dysfunction. Finally, we discuss current and future therapeutic approaches, including AAV-based gene therapy, RNA editing, X chromosome reactivation, and pharmacological interventions. Understanding the diverse functions and pathological mechanisms of MECP2 provides an important foundation for developing targeted therapies for Rett syndrome. Full article

Introduction: Elevated succinylacetone (SA) is the hallmark of tyrosinemia type 1, which requires immediate treatment. Mild SA elevation has also been recently reported in maleylacetoacetate isomerase deficiency (MAAID). Methods: We report on two cases of MAAID, review clinical features of MAAID and discuss its management. Results: Both cases displayed elevated SA and normal Tyrosine levels at newborn screening. Case 1 showed intermittent SA elevation; Nitisinone and dietary treatment were started, then discontinued after the identification of two variants in the GSTZ1 gene and the definitive diagnosis of MAAID. Case 2, showing no SA elevation at the confirmatory tests and two variants in the GSTZ1 gene, did not start treatment. mRNA analysis confirmed the pathogenicity of the c.68-12 G>A variant, found in both patients. Discussion: MAAID should be considered in newborns showing elevated SA and no variants in the FAH gene. Our study reports for the first time the course of SA in a patient affected by MAAID. Furthermore, it expands the molecular epidemiology of this rare disease, also investigating the pathogenicity of a novel splicing mutation. Although our data argue against medical treatment in MAAID, longer follow-up data are warranted. Full article

Background/Objectives: The most prevalent form of neurodegeneration with brain iron accumulation (NBIA) is pantothenate kinase-associated neurodegeneration (PKAN), caused by mutations in the PANK2 gene. The hallmark of PKAN is the “eye-of-the-tiger” sign, which is characterized by a bilateral region of central hyperintense signal surrounded by a hypointense signal in the medial globus pallidus on T2-weighted brain magnetic resonance imaging (MRI). Methods: Whole-exome sequencing (WES) was performed in four patients who presented with dystonia, cognitive impairment and abnormalities of the globus pallidus. All patients underwent comprehensive clinical and instrumental evaluations. Results: Molecular analysis using WES revealed PANK2 variants in all four cases. Two patients were homozygous for the known pathogenic variant c.1169A > T (p.N390I). The remaining two patients displayed compound heterozygotes, each carrying the novel splicing variant c.906-1G > A on one allele, combined with a different second variant on the other allele: the new missense variant c.617G > A (p.G206D) in one case and the known pathogenic variant c.1231G > A (p.G411R) in the other. In one case, brain imaging documented the transition from initial hyperintensity of the globus pallidus to the development of the “eye-of-the-tiger” sign. In two cases, MRI findings clearly demonstrated the characteristic “eye-of-the-tiger” appearance. Ultimately, in one case, the imaging likely captured a later disease stage, in which the “eye-of-the-tiger” sign was no longer visible, and only the residual hypointensity remained. Conclusions: This study describes two novel likely pathogenic variants and documents the full MRI progression of globus pallidus involvement in PKAN. The sequence starts with early T2 hyperintensity, followed by the emergence of the typical “eye-of-the-tiger” sign, and culminates in marked hypointensity in advanced stages. Since the initial clinical presentation may mimic mitochondrial disorders or other neurometabolic conditions, these imaging features are crucial for guiding differential diagnosis and enabling accurate disease identification. Full article

CD44, a structurally diverse cell-surface glycoprotein, plays a multifaceted and indispensable role in neural tissue across both physiological and pathological conditions. It orchestrates complex cell–extracellular matrix interactions and intracellular signaling through its variant isoforms and post-translational modifications and is broadly expressed in neural stem/progenitor cells, microglia, astrocytes, and selected neuronal populations. The interactions of CD44 with ligands such as hyaluronan and osteopontin regulate critical cellular functions, including migration, differentiation, inflammation, and synaptic plasticity. In microglia and macrophages, CD44 mediates immune signaling and phagocytic activity, and it is dynamically upregulated in neuroinflammatory diseases, particularly through pathways involving Toll-like receptor 4. CD44 expression in astrocytes is abundant during central nervous system development and in diseases, contributing to glial differentiation, reactive astrogliosis, and scar formation. Though its expression is less prominent in mature neurons, CD44 supports neural plasticity, circuit organization, and injury-induced repair mechanisms. Additionally, its expression at nervous system barriers, such as the blood–brain barrier, underscores its role in regulating vascular permeability during inflammation and ischemia. Collectively, CD44 emerges as a critical integrator of neural cell function and intercellular communication. Although the roles of CD44 in glial cells appear to be similar to those explored in other tissues, the expression of this molecule and its variants on neurons reveals peculiar functions. Elucidating the cell-type-specific roles and regulation of CD44 variants may offer novel therapeutic strategies for diverse neurological disorders. Full article

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease affecting approximately 1 million people in the United States. Despite extensive research into the mechanisms of disease development, many aspects of the biological changes during MS progression and the varying symptoms among patients remain unclear. In the era of high-throughput sequencing, transcriptome databases are flooded with data. However, bulk RNA sequencing (RNA-seq) data are typically used only for differential gene expression analysis. Alternative splicing, a key process that alters the transcriptome, can also be identified from bulk data. Here, we accessed 11 studies with bulk RNA-seq data of postmortem MS patients’ brain samples via NCBI’s Gene Expression Omnibus (GEO). We extracted additional information from these data by identifying exclusively alternatively spliced genes via replicate multivariate analysis of transcript splicing (rMATS) analysis. Our analyses revealed that changes in RNA splicing mediate distinct biological signals compared to those driven by differential gene expression. Gene ontology and protein do-main analyses of genes exclusively regulated by alternative splicing revealed distinct molecular differences between progressive and relapsing–remitting MS as well as among lesions from different brain regions and between white and gray matter. These findings highlight the critical role of alternative splicing and its associated pathways in MS disease development and progression. Full article

Background/Objectives: Therapeutic strategies for Neurofibromatosis Type I (NF1) that correct the underlying pathogenic NF1 variant hold promise for restoring neurofibromin function, reducing tumor burden, and improving patient outcomes by addressing the root cause of the disease rather than its symptoms. Beyond gene editing, transcript reprogramming via RNA trans-splicing has gained attention, particularly with the recent FDA approval of two trans-splicing-based drugs for IND phase 1/2a trials. This study tests whether trans-splicing group I intron ribozymes from Tetrahymena thermophila can be used to repair pathogenic variants of NF1 (pre-)mRNA by 3′-tail replacement. Methods: Splice sites on the NF1 mRNA were identified computationally and validated biochemically, and an efficiency-enhancing Extended Guide Sequence (EGS) of the corresponding ribozyme was identified in a combinatorial experiment. Results: The correct trans-splicing product of this ribozyme was validated in HEK293 NF1−/− cells expressing mNf1. Conclusions: This study established a splice site and activity-enhancing extended guide sequences for the repair of NF1 mRNA. Further optimization of the ribozyme, as well as improved delivery methods, may establish ribozyme-based RNA repair as a viable strategy for NF1 treatment. Full article

Sulforaphane (SFN) is a bioactive compound belonging to the isothiocyanate family, known for its neuroprotective properties. While transcriptomic studies have highlighted SFN’s role in regulating gene expression, its impact on alternative splicing (AS), a key regulatory mechanism in neuronal metabolism, remains underexplored. In this study, we investigated whether SFN pre-treatment influences mRNA splicing patterns in an in vitro neuronal model using retinoic acid (RA)-differentiated SH-SY5Y cells. Using a dedicated RNA-seq-based splicing analysis pipeline, we identified 194 differential alternative splicing events (DASEs) associated with SFN treatment. Gene Ontology enrichment revealed significant over-representation of DNA repair processes. To better understand the functional implications, we integrated in silico predictions of premature stop codons, DASE/miRNA hybridizations, and DASE/RNA-binding protein (RBP) motif occurrences. Our findings suggest that SFN may modulate splicing of key DNA repair genes, contributing to protecting neurons against DNA damage. These preliminary results underscore a novel layer of SFN’s molecular effects and propose it as a valuable adjuvant in physiological conditions to enhance cellular health. Further studies are warranted to dissect the mechanistic underpinnings of SFN-mediated AS and its relevance in DNA-damage-related disorders. Full article

Intron retention (IR) is one of the cellular mechanisms to perform alternative splicing and thus control gene expression in several mammalian cellular pathways. IR in PI-PLC γ1 mRNA was observed in some primary synoviocyte samples from osteoarthritis (OA) patients, likely due to inter-patient variability. The aim of the present manuscript was to explore the PI-PLC γ1 IR molecular mechanism as a consequence of nutraceutical treatment of synoviocytes and the molecular basis of individual response. To evaluate the gene expression modulation of molecules involved in mRNA splicing, an RNA-seq analysis was performed, and the transcription modulation of six differentially expressed genes was validated by RT-PCR. Moreover, through a silencing experiment, the relationship between PI-PLC γ1 IR and the six modulated genes was explored. Finally, two of them, the RNA-binding proteins CELF1 and PTBP3, whose mRNA levels were elevated in samples exhibiting IR, were analyzed in detail. CELF1 and PTBP3 were overexpressed in synoviocytes lacking PI-PLC γ1 IR, and we found that CELF1 was responsible for IR, whereas PTBP3 did not seem to be involved. In conclusion, in our experimental model, the role of CELF1 protein in PI-PLC γ1 IR was explored, opening new scenarios for understanding the molecular mechanisms underlying the IR phenomenon present in several kinds of diseases. Full article

For 40 years, Intron Retention (IR) was dismissed as splicing noise and is now recognized as a dynamic and evolutionarily conserved mechanism of post-transcriptional gene regulation. Unlike canonical splicing, which excises all introns from pre-mRNAs, IR selectively retains intronic sequences, albeit at seemingly random places; however, current research now reveals that this process is strategic in its retention. IR influences mRNA stability, localization, and translational potential. Retained introns can lead to nonsense-mediated decay, promote nuclear retention, or give rise to novel protein isoforms that contribute to expanding proteomic and transcriptomic profiles. IR is finely regulated by splice site strength, splicing regulatory elements, chromatin structure, methylation patterns, RNA polymerase II elongation rates, and the availability of co-transcriptional splicing factors. IR plays critical roles in cell-type and tissue-specific gene expression with observed patterns, particularly during neuronal, cardiac, hematopoietic, and immune development. It also functions as a molecular switch during cellular responses to environmental and physiological stressors such as hypoxia, heat shock, and infection. Dysregulated IR is increasingly associated with cancer, neurodegeneration, aging, and immune dysfunction, where it may alter protein function, suppress tumor suppressor genes, or generate immunogenic neoepitopes. Experimental and computational tools like RNA-seq, RT-PCR, IRFinder, and IntEREst have enabled transcriptome-wide detection and validation of IR events, uncovering their widespread functional roles. This review will examine current knowledge on the function, regulation, and detection of IR, and also summarize recent advances in understanding its role in both normal and pathophysiological settings. Full article