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At least 37 records · Page 2Linked to original sources
PubMed2026

CRISPR-mediated intronic knock-in of pre-amiRNA enables targeted gene silencing.

This study introduces an intronic artificial microRNA (IamiRNA) strategy that combines CRISPR-Cas9-mediated knock-in with endogenous miRNA processing for targeted gene silencing in plants. By inserting amiRNA precursors into introns of endogenous genes, this approach enables effective, tissue-specific gene silencing without persistent transgene expression, offering a promising tool for functional genomics and crop improvement.

Introns↗
PubMed2026

PRMT5-mediated intron retention triggers innate and adaptive immunity against cancer.

PRMT5 is expressed at high levels in many cancers, where it regulates diverse cellular pathways that contribute to oncogenesis. Here, we have defined a new role for PRMT5 in regulating and coordinating the interplay between the innate and adaptive immune response. This occurs, in part, through the influence of PRMT5 and E2F1 on RNA splicing and the presence of retained introns (RIs). We found that RIs have a propensity to form double-stranded RNAs that contribute to the innate response. Furthermore, many RIs contain non-canonical open-reading frames (ncORFs), which can be translated and then processed into small peptides that assemble with the MHC class I complex. Significantly, RI-derived peptides are highly immunogenic and, as a murine cancer vaccine, carrying a string of antigenic RI peptides, delayed tumour growth and enhanced survival. RIs are present in human tumour cells, and we identified T lymphocytes in human cancer patients, with antigen specificity for RI-derived peptides, that killed human tumour cells in vitro. Regulating intron retention thus offers a new therapeutic approach to enhance tumour immunogenicity.

Animals↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗
PubMed2026

X-linked spondyloepiphyseal dysplasia tarda misdiagnosed as growth hormone deficiency: identification of a novel intronic TRAPPC2 variant by whole-genome sequencing.

BACKGROUND: X-linked spondyloepiphyseal dysplasia tarda (SEDT) is a rare skeletal dysplasia caused by pathogenic variants in TRAPPC2 and typically presents in late childhood or adolescence with short-trunk disproportion and vertebral dysplasia. CASE PRESENTATION: We describe a family series centered on an adolescent male initially diagnosed with GHD due to reduced height velocity and subnormal GH stimulation results, who received recombinant human GH (rhGH) therapy for three years with negligible improvement. During puberty, he developed progressive short-trunk disproportion and characteristic radiographic features, including platyspondyly and posterior hump-shaped vertebral endplates, suggestive of SEDT. Whole-exome sequencing (WES) was nondiagnostic, whereas whole-genome sequencing (WGS) identified a novel intronic TRAPPC2 variant, c.239-20_239-12delinsAATGAA, initially classified as a variant of uncertain significance (VUS). Segregation analysis across the family enabled reclassification of the variant to likely pathogenic, confirming X-linked SEDT. The proband's younger brother exhibited earlier radiologic abnormalities and, notably, a favorable response to rhGH, whereas the younger sister-an asymptomatic heterozygous carrier-showed normal spinal morphology, consistent with expected female carrier phenotypes. CONCLUSIONS: This family-based report underscores the generally limited therapeutic effect of rhGH in SEDT while highlighting potential interindividual variability, as evidenced by the younger male sibling's response. It further emphasizes the diagnostic utility of WGS for detecting deep intronic variants missed by WES and the importance of segregation analysis in resolving VUS in rare skeletal dysplasias.

Humans↗
PubMed1978

Comparison of cloned rabbit and mouse beta-globin genes showing strong evolutionary divergence of two homologous pairs of introns.

Cloned beta-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most probably arose from a common ancestral sequence and diverged substantially during evolution.

Animals↗
PubMed1978

DNA methylation at a CCGG sequence in the large intron of the rabbit beta-globin gene: tissue-specific variations.

We have analysed DNA modification in a HapII site (CCGG) present in the major intron of the discontinuous rabbit beta-globin gene. In most somatic tissues, including erythroid and non-erythroid tissues, about 50% of the DNA is resistant to cleavage at this site by HapII, though 100% cleavage is found with the isoschizomer MspI. Since the former enzyme is unable to cleave CCGG sites if the internal C residue is 5-methyl C (and since methylation is the only form of CpG modification documented in animal DNA), while the latter enzyme cleaves DNA irrespective of methylation at this residue, we infer that 50% of the CCGG sites in the beta-globin gene intron are methylated in these tissues. The same site appears to be 100% methylated (judged by the same criterium) in sperm DNA and about 80% methylated in brain DNA. DNA from the rabbit SIRC cell line is entirely unmethylated at this site.

Animals↗
PubMed1979

Binding of mitochondrial ribosomal proteins to a mitochondrial ribosomal precursor RNA containing a 2.3-kilobase intron.

In Neurospora, the gene encoding the mitochondrial large subunit (25 S) rRNA contains an intervening sequence of approximately 2.3 kilobases. We have recently identified two temperature-sensitive, nuclear mutants (289-67 and 299-9) which are defective in splicing of the 25 S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants show decreased ratios of 25 S/19 S RNA and accumulate a novel 35 S RNA which appears to be a continuous transcript of the 25 S RNA gene, including the intervening sequence. In the present work, mitochondrial ribonucleoprotein particles present in the 50 S subunit peak from wild type and mutant 299-9 were analyzed by equilibrium centrifugation in CsCl gradients containing 25 mM MgCl2. The results show that 35 S RNA can be isolated as part of a ribonucleoprotein particle associated with nearly all of the large subunit ribosomal proteins. However, the particles appear to be less stable in CsCl gradients and more sensitive to nucleolytic degradation than particles derived from mature large subunits. Our results indicate that binding of ribosomal proteins to 35 S RNA could precede removal of the intron, but that removal of the intron may be required to achieve stable protein binding.

Base Sequence↗
PubMed2026

Intron editing of RISBZ1 confers thermotolerance for grain filling in rice.

RISBZ1 encodes the transcription factor bZIP58, which regulates grain filling; intron editing of RISBZ1 to eliminate aberrant alternatively spliced transcripts increased grain weight without compromising key agronomic traits under normal conditions, and enhanced grain weight and quality under heat stress conditions.

Oryza↗
PubMed2024

Identification of a novel intronic variant in COL4A2 gene associated with fetal severe cerebral encephalomalacia and subdural hemorrhage.

BACKGROUND: Genetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage. METHODS: Whole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant's effect. RESULTS: In this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del). CONCLUSION: Our study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

Female↗
PubMed2025

Prevalence of intronic repeat expansions in the RFC1 gene in Polish patients with cerebellar syndrome.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative ataxic disorder, which has been associated with intronic biallelic repeat expansions in the RFC1 gene. Our objective was to assess retrospectively the prevalence of CANVAS in Polish population. We screened 2523 Polish patients in whom other repeat expansions were excluded. To determine the repeat expansions in the RFC1 gene in patients, we performed RFC1-flanking PCR and repeat primed PCR (RP-PCR) and to measure the size of the expansion we used Southern blotting and optical genome mapping to compare the results. We have observed the biallelic pathogenic motif/unit AAGGG expansions in 4.6% and expansions of non-pathogenic motifs AAAAG, AAAGG in 25% patients of our studied population. This is the first large-scale cohort study that confirms the relatively frequent occurrence of the CANVAS in Polish population. To increase the current diagnostics of late-onset ataxias within an unexplained molecular background, we suggest involving the RFC1 repeat expansions analysis to the routine diagnostic workflow.

Humans↗
PubMed2026

Seventeen-year follow-up of hypophosphatasia diagnosed in middle-aged siblings harboring a novel intronic and a rare missense ALPL gene mutation.

Hypophosphatasia (HPP) is the rare inborn-error-of-metabolism that features impaired mineralization of the skeleton and teeth due to a deactivating mutation or mutations of the gene ALPL which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). We report 17-year follow-up of twin sisters and a brother referred in middle-age for painful proximal femoral "stress fractures" and then diagnosed with HPP. They reported generalized muscle and bone pain, metatarsal fractures, arthropathy and, since childhood, tooth loss. Their concordant findings were explained by compound heterozygosity in ALPL for a rare maternal missense mutation (c.1403C > T, p.Ala468Val) in exon 12, together with a novel presumably paternal change (c.863-14G > A) predicting a cryptic mRNA splice site in intron 8. Fractures continued during follow-up until one sister received a three-and-one-half-year course of hydroxyapatite-targeted TNSALP supplementation therapy (asfotase alfa) during which substantial improvement occurred in her clinical, biochemical, and functional parameters as well as quality of life. Following subsequent unplanned treatment cessation she suffered significant clinical deterioration, including new fractures and loss of mobility. Her bone histopathology documented osteomalacia. Treatment resumption restored its benefits. Among ten asymptomatic family members evaluated in this four-generation kindred, eight were carriers heterozygous for either ALPL mutation. Those harboring the maternal missense defect manifested mild hypophosphatasemia, suggesting a dominant-negative mutation effect. This experience underscores the importance of in-depth phenotyping and then clinical follow-up to characterize ALPL variant combinations, and for maintaining effective asfotase alfa treatment.

Humans↗
PubMed2025

Complex IV deficiency due to COX4I1 deep intronic and de novo variants results in progressive motor impairment and Leigh syndrome.

COX4I1 gene encodes cytochrome c oxidase subunit 4 isoform 1, involved in the early assembly stages of mitochondrial respiratory chain complex IV. To date, COX4I1 pathogenic variants have been reported in only a few cases, each exhibiting heterogeneous clinical phenotypes and limited functional data. Here, we describe the fourth reported case of COX4I1 deficiency associated with human disease, expanding the phenotypic and genetic spectrum of this rare mitochondrial disorder and providing novel clinical, molecular, and functional data. The herein reported individual presented with progressive deterioration of motor skills, intellectual disability and brain imaging abnormalities compatible with Leigh syndrome. Genetic studies combining short and long read next generation sequencing uncovered a peculiar genetic combination in this patient, harboring a de novo COX4I1 nonsense substitution in trans with an inherited deep intronic variant (c.[64C>T];[73+1511A>G]; p.[Arg22Ter];[Glu25ValfsTer9]). Functional studies performed in patient's tissues and transiently transfected cell lines demonstrated that the identified variants mainly exert their pathogenic effect by targeting COX4I1 protein levels, thereby impairing the proper assembly and activity of complex IV.Additionally, proteomic data in patient's fibroblasts suggested an underlying pathomechanism that involves not only the regulation of complex IV function but also the levels of mitoribosomal proteins. In summary, our findings shed light to clarify some of the main clinical features associated with COX4I1 deficiency and the molecular mechanisms involved in the pathogenesis of this disorder.

Humans↗
PubMed2025

Bayesian reconstruction and differential testing of excised introns.

MOTIVATION: Characterizing the differential excision of introns is critical for understanding the functional complexity of a cell or tissue, from normal developmental processes to disease pathogenesis. Most transcript reconstruction methods infer full-length transcripts from high-throughput sequencing data. However, this is a challenging task due to incomplete annotations and the heterogeneous expression of transcripts across cell-types, tissues, and experimental conditions. Several recent methods circumvent these difficulties by considering local splicing events, but these methods lose transcript-level splicing information and may conflate similar, but distinct transcripts. RESULTS: In this work, we formalize a new transcript reconstruction problem that interpolates between the full-length and local splicing perspectives by considering sequences of exon-exon junctions (SEEJs) that co-occur in transcripts. We then present a hierarchical Bayesian admixture model and posterior inference algorithms for computing SEEJs (BSEEJ), and a generalized linear model for characterizing differential SEEJ usage based on model parameter estimates. We show that BSEEJ achieves high F1 score for reconstruction tasks and improved accuracy and sensitivity in differential splicing when compared with six transcript and local splicing methods on simulated data. Lastly, we evaluate BSEEJ on experimental data based on transcript reconstruction, novelty of transcripts produced, model sensitivity to hyperparameters, and a functional analysis of differentially expressed SEEJs. AVAILABILITY AND IMPLEMENTATION: BSEEJ is freely available at https://github.com/bayesomicslab/BSEEJ.

Bayes Theorem↗
PubMed2026

A SINE-like insertion in intron 13 of the ATP7A gene is associated with a mild form of Menkes-like disease in a Cavalier King Charles Spaniel.

A 7-month-old intact male Cavalier King Charles Spaniel was presented for persistent glucosuria despite normoglycemia, failure to thrive, chronic diarrhea, and cerebellar ataxia. Fanconi syndrome was diagnosed, but the neurologic abnormalities were not fully explained. As a consequence of the early onset Fanconi syndrome, a hereditary process was suspected. Whole genome sequencing identified a private hemizygous SINE-like insertion into the ATP7A gene, at the end of intron 13, near the start of exon 14. In humans, variants in ATP7A are associated with Menkes disease, a disorder of copper metabolism associated with a spectrum of clinical signs including progressive neurodegeneration and connective tissue abnormalities. Clinically affected dogs with variants in ATP7A have not been reported previously. Although this case appears to represent a mild phenotypic presentation of Menkes-like disease, it raises the possibility that copper disorders aside from copper-associated hepatitis might exist in dogs. Further genetic screening and phenotypic characterization of rare genetic variants associated with copper metabolism would be beneficial to expand our knowledge of copper disorders in dogs and allow potential early intervention and modeling for metabolic diseases in humans.

Animals↗
PubMed2025

Expression of intron-containing HIV-1 RNA induces NLRP1 inflammasome activation in myeloid cells.

Despite the success of antiretroviral therapy in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1), persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells, such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses, demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report, we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1β secretion in macrophages and microglia in an RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1β secretion, which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1β secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV-infected macrophages, overexpression of NLRP1 significantly enhanced IL-1β secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA, but not multiply-spliced HIV-1 RNA, with NLRP1, suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together, these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV-infected myeloid cells.

HIV-1↗
PubMed1978

Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries.

Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ov-mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at exon-intron boundaries, the nucleotide sequence alone cannot define unique excision-ligation points for the processing of a possible ov-mRNA precursor. However, the sequences in these boundary regions share common features; this leads to the proposal that there are, in fact, unique excision-ligation points common to all boundaries.

Animals↗