pre-mRNA Splicing Research Articles

Objective To investigated the pathogenic mechanism of NFIX frameshift mutations in Malan syndrome. Methods Reviewed the clinical diagnosis and treatment processes of the Malan syndrome proband, analyzing the relationship between NFIX frameshift mutation genotypes and clinical phenotypes, and the inheritance pattern. To analyzed the functional domain where the mutation was located and the conservation of the mutated amino acid residue, thereby elucidating the potential impact of the mutation on the protein. Validated effects on pre-mRNA splicing using RDDC SC , SpliceAI, and FF databases. Assessed variant pathogenicity via MutationTaster, PolyPhen-2, and VarCards. Constructed wild-type/mutant plasmids, transfected to HEK293T cells, and quantified NFIX mRNA and protein expression levels via qPCR and Western blot. Analyzed degradation pathways using ubiquitin-proteasome inhibitor MG132 and autophagy-lysosome inhibitor Chloroquine (CQ). Results The proband exhibited intellectual disability, distinctive facial features, ocular abnormalities, scoliosis, and primary infertility. A de novo mutation in NFIX (c.164delC, p.Ala55Glyfs*2) associated with these phenotypes was identified. Neither the proband’s father nor his mother was found to have this mutation. Parental testing confirmed de novo inheritance. The amino acid at position 55 was highly conserved and had been Alanine in 5 species. Results from databases including RDDC SC , SpliceAI, and FF indicated that the NFIX c.164delC p.Ala55Glyfs*2 mutation did not affect splicing function. Predictions by MutationTaster and PolyPhen-2 classified the c.707G>A p.Arg236Gln mutation as “damaging,” suggesting an altered amino acid sequence, frameshift mutation, NMD, and potential modification of protein characteristics. Quantitative real-time PCR (qPCR) analysis detected comparable mRNA levels between mutant and wild-type strains. In contrast, Western blotting revealed significantly diminished protein expression in the mutant ( P < 0.05), suggesting post-transcriptional regulation effects. Results from protein degradation pathway analysis demonstrated that the truncated protein generated after mutation was degraded via the ubiquitin-proteasome pathway. Conclusion The NFIX c.164delC p.Ala55Glyfs*2 frameshift mutation did not significantly affect mRNA expression levels, but induced protein degradation via the ubiquitin-proteasome pathway, resulting in haploinsufficiency and ultimately causing Malan syndrome.

Circular RNAs (circRNAs) have emerged as key regulators in human diseases, yet their mechanisms of action in chronic obstructive pulmonary disease (COPD) remain largely unknown. In this study, the conserved mammalian circRNA circFCHO2 was shown to play critical roles in COPD. The expression level of circFCHO2 was significantly increased in COPD cell models, mouse models, and human lung tissue samples. Moreover, we demonstrated that circFCHO2 promotes epithelial‒mesenchymal transition (EMT) in bronchial epithelial cells and extracellular matrix (ECM) remodeling. Mechanistically, circFCHO2 binds to and facilitates the nuclear translocation of PTBP1, thereby inhibiting the splicing of GRN pre-mRNA, which reduces PGRN protein expression levels and activates the NF-κB pathway. This activation of the NF-κB signaling pathway regulates the expression of EMT and ECM remodeling-related proteins, leading to the occurrence of airway remodeling. circFCHO2 knockdown reverses cigarette smoke-induced emphysema and airway remodeling of COPD in mice. Overall, our study advanced the understanding of the molecular mechanisms by which circRNAs contribute to airway remodeling in COPD patients.

Low phosphorus (P) use efficiency of rice, in spite of sufficient P present in soil (mainly as insoluble complex), and excessive use of phosphatic fertilizers by farmers may cause several issues including higher cost of cultivation and environmental pollution. Alternative splicing of pre-mRNA is one of the strategies for transcriptome/proteome diversity that help plants to optimize acquisition and mobilization of P. Comparative analysis of rice [Pusa-44 and its Near-Isogenic Line (NIL)-23] grown hydroponically under contrasting conditions (16 ppm and 0 ppm) until vegetative stage deciphered stress-induced alternative splicing regulated through Pup1 QTL. The modulation in alternative splicing in root and shoot of rice under stress can be attributed to the regulatory function of Pup1. In addition to a considerable increase in the number of genes showing up-regulated expression, ∼20 genes demonstrated differential expression as well as alternative splicing (with a decrease in root but increase in shoot) in NIL-23under stress. Increase in alternative splicing of transcripts for ‘iron-regulated bHLH transcription factor’, ‘regulation of P homeostasis’ in roots while ‘serine hydroxymethyltransferase’ and ‘iron-regulated bHLH transcription factor’ in shoot, particularly those associated with P homeostasis/P-starvation stress-responsive signaling pathway, were recorded in the case of NIL-23 under stress. Genes/transcripts, particularly those associated with biological process in roots, showing reversed (increased vs. decreased) level of splicing in NIL-23 (compared to that in its parent) on P-starvation confirmed regulatory role of the QTL. Better understanding of splicing event/gene/transcript and its regulation might provide novel strategies to modulate gene expression for nutrient homeostasis and enhancing stress resilience in crop plants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07379-9.

Telomeres perform multiple functions to maintain genome stability, including telomere length regulation, chromosome end protection, and meiotic chromosome dynamics. These functions are governed by shelterin, a telomere-binding protein complex. Here, we show that Tls1 and Cay1 act at distinct steps in pre-mRNA splicing, specifically ensuring sufficient protein levels of Schizosaccharomyces pombe shelterin components Rap1 and Poz1, which are critical for telomere maintenance. Accordingly, deletion of tls1+ and cay1+ synergistically reduced Rap1 and Poz1 protein levels. Analyses of the phenotypes of single and double tls1∆ and cay1∆ mutants indicated that different telomere functions vary in their dependence on Rap1 levels: telomere length regulation and, to a lesser extent, meiosis require higher protein abundance, whereas chromosome end protection can be sustained with minimal amounts. These findings reveal a hierarchical requirement for Rap1 across telomere functions.

The HGD gene encodes homogentisate 1,2-dioxygenase. A deficiency of this enzyme causes alkaptonuria (AKU; OMIM 203500), a monogenic autosomal recessive metabolic disorder. The global incidence of alkaptonuria is estimated at 1 in 250,000 to 1,000,000 live births. A large number of pathogenic nucleotide variants disrupt pre-mRNA splicing, leading to hereditary diseases. Many potentially splice-disruptive variants, including those in coding regions, remain uncharacterized. This lack of data makes clinical interpretation more difficult and can complicate diagnosis. We systematically analyzed 27 HGD variants predicted to affect splicing. Candidate variants from public databases (ClinVar, HGDdatabase) and our patient cohort were prioritized using in silico splicing predictions and evaluated with a minigene splicing assay in HEK293T cells. Based on the obtained functional analysis data, the variants were reclassified according to ACMG/AMP guidelines. In total, 13 variants changed their classification (9 were upgraded and 4 were downgraded), while 5 variants retained their pathogenicity class after analysis. Ten missense/nonsense variants were not reclassified, as no significant splicing disruption was detected. These findings improve the pathogenicity assessment of HGD variants, support more accurate diagnosis, and lay the foundation for future therapeutic strategies targeting splicing defects in AKU.

As a major contributor to cancer-associated deaths, advanced colorectal cancer (CRC) has a constrained range of effective treatment options. The short isoform of bromodomain-containing protein 4 (BRD4-S) has recently been implicated as a potential oncogenic driver; however, its regulatory mechanisms and functional role in CRC remain incompletely understood. BRD4-S expression, regulation, and function in CRC were investigated through bioinformatics analyses of the Cancer Genome Atlas (TCGA) datasets, in vitro studies using CRC cell lines (HT29, SW620), and in vivo xenograft models in nude mice. Experimental approaches included quantitative real-time PCR (qRT-PCR), Western blotting, co-immunoprecipitation, RNA immunoprecipitation, immunofluorescence, colony formation, Cell Counting Kit-8 (CCK-8), and scratch assays. Gene enrichment and interaction analyses were performed to identify relevant pathways and molecular partners. BRD4-S was markedly upregulated in CRC tissues and cell lines, and elevated BRD4-S expression correlated with poorer patient survival. Silencing BRD4-S, but not BRD4-L, significantly impaired CRC cell proliferation, migration, and tumor growth in vivo. Mechanistically, the RNA helicase DEAD-box helicase 27 (DDX27) interacted with Serine and Arginine Rich Splicing Factor 6 (SRSF6) to promote alternative splicing of BRD4 pre-mRNA toward the BRD4-S isoform. Inhibition of SRSF6 phosphorylation suppressed BRD4-S production and blocked activation of the mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases ERK signaling pathway, identified as a key downstream effector of BRD4-S. This study defines a novel DDX27-SRSF6-BRD4-S-MAPK/ERK signaling axis that drives CRC progression. These findings underscore the therapeutic potential of targeting BRD4 isoform switching and its regulatory splicing machinery in CRC.

Pseudouridine (Ψ) is a widespread RNA modification in various RNA species, including rRNA, tRNA, snRNA and mRNA. Ψ plays a crucial role in RNA metabolism, where it regulates pre-mRNA splicing and affects protein translation. Whether and how Ψ may regulate transcription have not been adequately studied. Here, we report that pseudouridine synthase 7 (PUS7) can mediate pseudouridylation of 7SK small nuclear RNA (snRNA), a regulator of RNA polymerase II (Pol II) promoter-proximal pausing. PUS7 loss leads to hypo-pseudouridylation of 7SK, which promotes dissociation of the positive transcription elongation factor b (P-TEFb) complex from 7SK. The release of P-TEFb from 7SK increases serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and enhances transcription elongation. In colorectal cancer (CRC) cells, the Ψ level of 7SK can be modulated by PUS7, or by site-specifically targeted pseudouridylation through dCas13b-guided system. Hypo-pseudouridylation on 7SK upon PUS7 depletion promotes KLF6/DDIT3-mediated cell apoptosis and sensitizes CRC cells to 5-FU.

DNA topoisomerase II promotes N6-adenosine mRNA methylation.

Alterations in mRNA splicing play a critical role in driving the molecular heterogeneity of many cancers, including urothelial carcinoma, by contributing to disease progression, treatment response, and clinical outcomes. These splicing changes can arise from somatic mutations in core spliceosomal components or through alternative splicing events affecting cancer-associated genes. In this review, we examine how dysregulation of pre-mRNA splicing influences key aspects of urothelial carcinoma biology, including cell proliferation, invasion, metastasis, modulation of the immune microenvironment, metabolism, and therapeutic resistance. We highlight frequently observed splicing-factor mutations and discuss the impact of aberrant splicing and cancer-specific isoforms on prognosis. We also explore splicing alterations associated with susceptibility to urothelial carcinoma and review emerging therapeutic strategies, such as splice-switching oligonucleotides and small molecule spliceosome inhibitors, that offer promising avenues for precision medicine in this disease.

Abstract Background and Aims Glycosphingolipid accumulation is a common feature of Anderson Fabry disease (AFD), a lysosomal storage disorder caused by deficient activity of α-galactosidase A encoded by the GLA gene. Over 1000 mutations in the GLA gene have been described, including missense and nonsense mutations, rearrangements and splicing defects, which have been identified as causes of AFD. The GLA gene has seven splice variants, and aberrant splicing accounts for approximately 5% of AFD mutations, but information on pre-mRNA splicing is available for only a limited number of patients. Many polymorphic GLA variants have been described, but it is unclear whether complex intronic haplotypes (CIH) formed by combinations of these variants are associated with disease onset, although several studies report cases of patients with classical forms of AFD with only haplotypes. We present a case of Fabry disease in a man with the CIH mutation and classic manifestations. Case presentation A 64-year-old male patient was admitted to our nephrology unit because of an elevated serum creatinine level (sCr 1.8 mg/dL) and proteinuria of 2 g/24 h. The anamnesis reported a previous diagnosis of hypertension at the age of 50 years treated with antihypertensive drugs, episodes of nephrolithiasis, sensorimotor peripheral polyneuropathy evaluated by electromyography in the lower limbs and, in the last year, hands acroparesthesias. The patient was ineligible for percutaneous kidney biopsy for bilateral parapelvic and voluminous left polar cysts. In addition, immunological evaluations reported antinuclear antibody (ANA) positivity (1:320), negative serum and urine immunofixation, normal free light chains ratio (dFLC), normal C3 and C4, normal serum immunoglobulin levels (IgG-IgM-IgA), and glycosylated Hb. A cardiac left hypertrophy (increase in SIV and PP) and mitral/aortic valve insufficiency was demonstrated by echocardiography and cardiac RMN. Based on the previous clinical data and a family history of CKD and cardiovascular disease (mother died of ischaemic stroke), we decided to test for Fabry disease. Method DNA was extracted from dried blood spots and sequenced for all GLA exons and up to 50 base pairs upstream and downstream of the intron-exon boundaries at both sites. Total RNA was extracted from peripheral blood mononuclear cells (PBMC) for functional haplotype characterization (CIH). The RNA was reverse transcribed into cDNA and the mRNA expression was analyzed by qPCR. Total proteins were extracted from the PBMCs and the expression of alpha-Gal protein was determined by Western blotting analysis. Results Genetic analysis revealed the presence of 3 variants within (CIH): c.370-81_370-77delCAGCC, c.640-16 A > G, and c.1000-22 C > T. In addition, qPCR results show a higher expression of GLA mRNA levels in the patient compared to a healthy subject (control) (Fig. 1), suggesting a possible activation of cryptogenic splicing. Conversely, western blot analysis revealed lower alpha-Gal protein expression in the patient compared to the control (Fig. 2). This could explain the clinical Fabry phenotype in our patient. Conclusion Thus, our clinical case reports a specific genetic combination of SNPs in a patient with a multisystemic clinical alteration that, according to biomolecular analysis, could be associated with alpha-Gal protein deficiency. Although we cannot consider these mutations as classical AFD genetic features and further studies are needed to elucidate how inheritance of complex intronic haplotypes affects GLA regulatory mechanisms, genetic analysis of the entire GLA gene sequence and multiple ligation dependent probe amplification (MLPA), the study of GLA expression could be extremely useful tools to explain the clinical phenotype of these patients and define a possible treatment. Moreover, our clinical report demonstrated the importance of strengthening the collaborative network between clinicians and other technical figures (genetists, biologists) in our nephrology centres to better diagnose and treat fragile patients with rare kidney diseases.

RNAs engage diverse protein partners and localize to specific subcellular compartments, yet dissecting proteomes associated with low-abundance or dispersed RNA molecules remains a challenge. We present an enhanced hybridization-proximity labeling (HyPro) technology for in situ proteome profiling of endogenously expressed RNA microcompartments. We re-engineer the HyPro enzyme and optimize proximity biotinylation conditions to identify proteins associated with compact RNA-containing nuclear bodies, small pre-mRNA clusters, and individual transcripts. Applying this approach to pathogenic G4C2 repeat-containing C9orf72 RNAs, retained as single-molecule foci in the nuclei of amyotrophic lateral sclerosis (ALS) patient-derived pluripotent stem cells, we reveal extensive interactions with disease-linked paraspeckle markers and a specific set of pre-mRNA splicing factors. These findings highlight early RNA processing and localization defects in ALS that may contribute to this late-onset neurodegenerative disorder. Overall, HyPro provides a broadly applicable platform for mapping RNA-protein interactions, enabling insights into RNA biology and its dysregulation in disease.

Heterogeneous nuclear ribonucleoprotein M (hnRNPM) is an RNA-binding protein that is aberrantly expressed in tumorigenesis. However, the alternative splicing regulation by hnRNPM in human colorectal cancer (CRC) remain unclear. Herein, we observed that hnRNPM was highly expressed in CRC tissues. The knockdown of hnRNPM inhibited the proliferation of colon cancer cells both in vivo and in vitro. Using RNA-seq, we screened and identified several alternative splicing events regulated by hnRNPM. Knockdown of PLEKHB2-S splice isoform could reduce the growth of colon cancer cells in vitro and in vivo, predicting its role in malignant proliferation of colon cancer cells. Mechanically, the minigene reporter assay indicated the predominant regulatory roles of hnRNPM in PLEKHB2 splicing. The in vivo crosslinking immunoprecipitation (CLIP) assay demonstrated the direct binding of the RNA recognition motif RRM2 of hnRNPM protein to exon 9 of PLEKHB2 pre-mRNA. HnRNPM facilitated the skipping of alternative exon 8 in PLEKHB2 by binding to the constitutive exon 9. Furthermore, we developed cationic microbubbles shRNA/CMBs and transferred to colon cancer cells via ultrasound-targeted microbubble disruption (UTMD). The size and zeta potentials of CMBs and shRNA-CMBs were measured and the optimal concentration range of shRNA/CMBs were screened with low cytotoxicity. The introduction of sh-hnRNPM/CMBs or sh-PLEKHB2-S/CMBs suppressed the proliferation of colon cancer cells in vitro and in vivo. Collectively, our findings identify that hnRNPM dysregulates colorectal carcinoma proliferation at the molecular level of splicing regulation and predicate sh-hnRNPM/CMBs and its splicing target sh-PLEKHB2-S/CMBs as promising therapeutic drugs and innovative strategies for treating colorectal cancer. Schematic diagram of the functions of UTMD mediated sh-hnRNPM/CMBs in colorectal cancer and emerging mechanism by which hnRNPM promotes the malignant proliferation of CRC by regulating the alternative splicing of PLEKHB2 pre-mRNA. The picture was created with BioRender.com.

Age-related macular degeneration (AMD), a leading cause of vision loss in elderly individuals, is a multifactorial disease driven by genetic, environmental, and cellular aging processes. Emerging evidence highlights the critical role of ribonucleic acid (RNA) splicing dysfunction in AMD pathogenesis, with a focus on the U1 small nuclear ribonucleoprotein (U1 snRNP) complex, a key spliceosome component. U1 snRNPs ensure the fidelity of RNA cotranscription and pre-mRNA splicing initiation, and their dysfunction has been implicated in neurodegenerative disorders and other age-related diseases. This narrative review explores the impact of U1 snRNP dysregulation on retinal cells, focusing on its role in transcriptomic instability, impaired protein homeostasis, cellular stress, impaired autophagy, and inflammation, which are important features of AMD pathogenesis. Finally, we propose that targeting U1 snRNP dysfunction could provide a novel therapeutic approach to slow, prevent, or restore retinal degeneration, offering insights into broader implications for age-related diseases. Understanding the molecular mechanisms underlying U1 snRNP dynamics in retinal health and degeneration is essential for developing innovative and effective treatments for AMD, which may provide ways to delay or reverse the effects of aging and associated diseases.

BackgroundHemophilia A is a rare, severe X-linked recessive inherited hemorrhagic disorder caused by F8 gene dysfunction, which is characterized by spontaneous or post-traumatic bleeding tendencies. The pathogenic variants identified in the F8 gene contribute to prenatal diagnosis and genetic counseling services for patients and their families.MethodsWe used inverse shifting-PCR (IS-PCR), direct DNA sequencing, bioinformatics predictions, cDNA sequencing, and minigene splicing assays to explore candidate variants in a Chinese family with hemophilia A. The identified variant was classified in accordance with ACMG/AMP guidelines.ResultsA novel c.6115+5_6115+6delinsAG variant at 5′ splice sites (5’ss) in exon 19 was identified in a 14-year-old Chinese boy with hemophilia A by DNA sequencing, which is inherited from his asymptomatic carrier mother. Multiple bioinformatics prediction tools, including SD-Score, information content (Ri), varSEAK, and RDDC RNA splicer, predicted that this variant might affect the normal pre-mRNA splicing. Both cDNA sequencing and minigene splicing assays proved that the variant led to exon 19 skipping in the F8 gene, which was ultimately classified as pathogenic according to the ACMG/AMP guidelines.ConclusionThe c.6115+5_6115+6delinsAG variant in the F8 gene is considered to be responsible for hemophilia A in this family. This dinucleotide variant located at 5’ss of the gene is initially reported. Our study has expanded the mutation spectrum of F8 and provided a basis for prenatal and clinical diagnosis.

Pre-mRNA splicing is catalyzed by the ribonucleoprotein (RNP) complex known as the spliceosome. The spliceosomes are dynamic and undergo constant rearrangement, leading to the formation of the different spliceosomal complexes A, B, Bact, C, C*, and P. Isolation of the spliceosomal complex at a specific intermediate stage requires a means to enrich it. This study describes a strategy for studying intermediate spliceosomal complexes by combining BioID with splicing assays. The MINX splicing substrate with a mutation at the 3′ splice site was utilized to arrest and capture the spliceosomal C* complex before the second catalytic step of splicing. The splicing substrate also contains binding sites for the MS2 coat protein, which facilitates the pull-down of assembled complex by FLAG-MS2-tagged RNP immunoprecipitation and determines the captured proximal proteins by mass spectrometry.

Aging is a risk factor for atrial fibrillation (AF). In 19-month-old mice, increases in AF inducibility are associated with enhanced protein levels of fat mass and obesity-associated protein (Fto), and reduced N6-methyladenosine (m6A) modification in atrial tissue. Whether Fto-regulated m6A demethylation is involved in aging-induced AF remains unclear. AF inducibility and electrophysiology were performed through programmed stimulation and optical mapping. The intensities of slow delayed rectifier potassium currents (IKs) were measured by patch-clamp. m6A-sequencing revealed that Kcne1 mRNA was m6A-demethylated in aging mouse atria. Kcne1 knockdown in 2-month-old mice increased AF inducibility. Aging mice with cardiomyocyte-specific Fto knockout had increased Kcne1 mRNA and protein levels, with reduced susceptibility to AF. Additionally, overexpression of wild-type Fto, rather than a catalytically inactive mutant in 2-month-old mice, reduced Kcne1 protein levels, leading to enhanced IKs current and AF inducibility. Furthermore, the negative relationship between FTO and KCNE1 was confirmed in left atrial appendage samples from AF patients. In iPSC-derived atrial cardiomyocytes, FTO-mediated KCNE1 demethylation repressed KCNE1 pre-mRNA splicing, mRNA nuclear export, and translational efficacy. Collectively, aging-induced elevation of Fto represses m6A methylation of Kcne1, which in turn leads to reductions in Kcne1 mRNA and protein levels in atrial cardiomyocytes, thereby increasing AF inducibility.

Antisense oligonucleotides (AONs) are small pieces of chemically modified DNA or RNA that bind to RNA in a sequence-specific manner based on Watson-Crick base-pairing. Splice-switching AONs are designed to modulate pre-mRNA splicing, thereby for instance restoring protein expression or modifying the eventual protein to restore its function or reduce its toxicity. Given the current lack of in silico methods that adequately predict off-target splicing events, assessment of off-target effects of AONs in human cells using RNAseq could be a promising approach. The identification and prioritization of potential off-target effects for validation and further investigation into the biological relevance would contribute to the development of safe and effective AONs. In this study, we used three different splice-switching AONs targeting three different human transcripts to study their transcriptome-wide, hybridization-dependent off-target effects with short read RNAseq. Using the computational tools rMATS and Whippet, we identified differential splicing events of which only a minority could be explained by hybridization, illustrating the difficulty of predicting off-target effects based on sequence homology. The main splicing events could all be validated with RT-PCR. Furthermore, from the three AONs studied, one AON induced considerably more changes in gene expression and splicing compared to the two other AONs assessed, which was confirmed in a validation experiment. Our study demonstrates that interpretation of short read RNAseq data to determine off-target effects is challenging. Nonetheless, valuable results can be obtained as it allows the comparison of toxicity between different AONs within an experiment and identification of AON-specific off-target profiles.

Y‑box binding protein 1 (YBX1/YB‑1) is a DNA/RNA‑binding protein, which plays a crucial role in promoting tumor progression and resistance to anticancer drugs. YBX1 is widely involved in a range of biological processes such as DNA repair, mRNA transcription, pre-mRNA splicing, mRNA stability regulation, translation and exosome sorting. In addition to these canonical DNA/RNA‑binding protein functions, YBX1 can also have a regulatory role in N6‑methyladenine (m6A) and 5‑methylcytosine (m5C) RNA modification. Moreover, YBX1 functions as an m5C reader to regulate mRNA stability, thus modulating gene expression and affecting disease development. Furthermore, SU056, an inhibitor of YBX1, has been shown to reverse drug resistance and prevent tumor development. In the present review, the structure of YBX1 and its functions in RNA methylation modifications are summarized, and its effects on m5C and m6A RNA modifications in cancer progression and drug resistance are emphasized.

BackgroundHub1, a conserved ubiquitin-like protein, is essential for pre-mRNA splicing and transcriptional regulation in Saccharomyces cerevisiae. Despite its known functions, the genome-wide effects of Hub1 overexpression remain largely uncharacterized. This study investigates the transcriptomic and splicing landscape changes triggered by Hub1 overexpression using an integrative bioinformatic approach.ResultsWe analyzed RNA-seq data from the GSE84215 dataset, employing differential expression, alternative splicing, functional enrichment, and network-based methods. DESeq2 identified 3,915 differentially expressed genes (DEGs; 1,964 upregulated, 1,951 downregulated, padj < 0.05), demonstrating extensive transcriptional reprogramming. Principal component analysis revealed that Hub1 overexpression explained 98% of transcriptional variance, indicating its dominant regulatory influence. Using rMATS, we detected seven exon skipping events, with DYN2 showing significant differential splicing (FDR = 0.0481, ΔPSI = − 0.036). MaxEntScan analysis confirmed that DYN2’s 5′ splice site is significantly weaker than canonical yeast splice sites (score = − 18.32, p = 0.03), consistent with Hub1’s role in facilitating non-consensus splicing. Functional enrichment analyses revealed metabolic reprogramming, with upregulated pathways including biosynthesis of secondary metabolites and carbon metabolism, while growth-related processes like ribosome biogenesis and cell cycle were downregulated. Gene Set Enrichment Analysis (GSEA) further supported stress response activation (p53 signaling, NES = 1.255) and cell cycle suppression (NES = − 0.692). Weighted Gene Co-expression Network Analysis (WGCNA) identified 61 co-expression modules, with the brown module highly correlated with Hub1 overexpression (r = 0.99, p < 0.001) and enriched in biosynthetic and proteasome pathways. Protein-protein interaction network analysis revealed 35 Hub1 interactors, including spliceosomal components, reinforcing its central role in RNA processing.ConclusionOur findings reveal that Hub1 overexpression drives coordinated transcriptional and post-transcriptional changes, promoting metabolic reprogramming while specifically modulating splicing of genes with weak splice sites like DYN2. These results establish Hub1 as a dual regulator linking transcriptional control with splicing precision, suggesting a regulatory mechanism that enhances cellular adaptability under stress conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12006-w.

Abstract Reproduction is a key determinant of efficiency and economic sustainability in beef cattle operations, as it directly influences calf production. In beef herds, where reproductive management is generally less intensive than in dairy systems, many pregnancy losses (PL) go undetected, making it challenging to identify their underlying causes. Understanding the genetic basis of PL is crucial for enhancing reproductive success and improving breeding strategies. The objective of this study was to estimate variance components and investigate genetic factors associated with PL in Brahman cattle. Phenotypic records consisted of 29,905 pregnancy and abortion records from nulliparous, primiparous, and multiparous cows raised in two herds in Bolivia between 1998 and 2021. Both herds followed reproductive disease prevention protocols. Pregnancy was confirmed by rectal palpation approximately 60 days after the breeding season ended, based on cows not returning to heat. Cows with successful pregnancies were assigned a phenotype of 1, while cows experiencing pregnancy loss were assigned a phenotype of 2. A total of 921 animals were genotyped using a medium-density SNP chip (~52K markers). Variance components were estimated using a threshold repeatability model to assess the binary response to PL through a single-step genomic BLUP procedure. SNP quality control excluded markers with unknown positions, those on sex chromosomes, MAF &amp;lt; 0.05, Hardy-Weinberg disequilibrium (P &amp;lt; 10⁻⁶), low call rates (&amp;lt; 90%), and excess heterozygosity. Genomic regions and candidate genes were identified based on the percentage of genetic variance explained by 10 adjacent SNP across the entire bovine genome. The Ensembl Biomart tool with the Genes 113 database and bovine genome ARS-UCD1.3 dataset was used to identify the gene content of genomic regions. Orthologous genes had their functions investigated in MeSH (Medical Subject Headings), OMIM and OMIA. Orthologous genes were also identified in humans (H. sapiens), rats (R. norvegicus), and mice (M. musculus). The heritability estimate for PL was low (0.114), but the presence of genetic variance suggests that selection for improved reproductive performance is feasible. Genome-wide association analyses identified 17 candidate regions in 12 chromosomes containing 92 genes. CDC40 (BTA9) is involved in pre-mRNA splicing and cell cycle regulation, with potential links to reproductive inefficiencies. DIS3 (BTA12) plays a crucial role in RNA metabolism, and its mutations are associated with embryonic lethality and infertility. MKS1 (BTA19) is essential for ciliogenesis, with mutations linked to severe congenital abnormalities and early embryonic death. The candidate regions reported in this study provide insights into the genetic architecture of PL for identifying and selecting animals with improved reproductive performance, ultimately enhancing the productivity of Brahman cattle. Moreover, our findings contribute to a better understanding of the genetic and physiological mechanisms underlying pregnancy retention in beef cattle.