Intronic Site Research Articles

Different germline variants in the XPA gene are associated with severe, intermediate, or mild neurodegeneration in xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease caused by pathogenic variants in seven nucleotide excision repair genes (XPA to XPG) and POLH involved in translesion synthesis. XP patients have a >1000-fold increased risk for sunlight-induced skin cancers. Many Japanese XP-A patients have severe neurological symptoms due to a founder variant in intron 3 of the XPA gene. However, in the United States we found XP-A patients with milder clinical features. We developed a simple scoring scale to assess XP-A patients of varying neurological disease severity. We report 18 XP-A patients examined between 1973 and 2023 under an IRB approved natural history study. Using our scale, we classified our XP-A cohort into severe (n = 8), intermediate (n = 5), and mild (n = 5) disease groups at age 10 years. DNA repair tests demonstrated greatest reduction of DNA repair in cells from severe patients as compared to cells from mild patients. Nucleotide sequencing identified 18 germline pathogenic variants in the 273 amino acid, 6 exon-containing XPA gene. Based on patient clinical features, we associated these XPA variants to severe (n = 8), intermediate (n = 6), and mild (n = 4) clinical phenotypes in the patients. Protein structural analysis showed that nonsense and frameshift premature stop codon pathogenic variants located in exons 3 and 5 correlated with severe disease. Intermediate disease correlated with a splice variant at the last base in exon 4. Mild disease correlated with a frameshift variant in exon 1 with a predicted re-initiation in exon 2; a splice variant that created a new strong donor site in intron 4; and a large genomic deletion spanning exon 6. Our findings revealed correlations between disease severity, DNA repair capacity, and XPA variant type and location. In addition, both XPA alleles contributed to the phenotypic differences in XP-A patients.

BCAS2 and hnRNPH1 orchestrate alternative splicing for DNA double-strand break repair and synapsis in meiotic prophase I

Understanding the intricacies of homologous recombination during meiosis is crucial for reproductive biology. However, the role of alternative splicing (AS) in DNA double-strand breaks (DSBs) repair and synapsis remains elusive. In this study, we investigated the impact of conditional knockout (cKO) of the splicing factor gene Bcas2 in mouse germ cells, revealing impaired DSBs repair and synapsis, resulting in non-obstructive azoospermia (NOA). Employing crosslinking immunoprecipitation and sequencing (CLIP-seq), we globally mapped BCAS2 binding sites in the testis, uncovering its predominant association with 5’ splice sites (5’SS) of introns and a preference for GA-rich regions. Notably, BCAS2 exhibited direct binding and regulatory influence on Trp53bp1 (codes for 53BP1) and Six6os1 through AS, unveiling novel insights into DSBs repair and synapsis during meiotic prophase I. Furthermore, the interaction between BCAS2, hnRNPH1, and SRSF3 was discovered to orchestrate Trp53bp1 expression via AS, underscoring its role in meiotic prophase I DSBs repair. In summary, our findings delineate the indispensable role of BCAS2-mediated post-transcriptional regulation in DSBs repair and synapsis during male meiosis. This study provides a comprehensive framework for unraveling the molecular mechanisms governing the post-transcriptional network in male meiosis, contributing to the broader understanding of reproductive biology.

A germline PDGFRB splice site variant associated with infantile myofibromatosis and resistance to imatinib

Infantile myofibromatosis is characterized by the development of myofibroblastic tumors in young children. In most cases, the disease is caused by somatic gain-of-function variants in platelet-derived growth factor receptor beta (PDGFRB). Here, we report a novel germline intronic PDGFRB variant, c.2905-8G>A, in six unrelated infants with multifocal myofibromatosis and their relatives. We performed constitutional and tumor DNA and RNA sequencing to identify novel variants, which were subsequently characterized in cellular assays. All patients had multiple skin nodules, four had bone lesions and two had aggressive disease with bowel obstruction. The c.2905-8G>A substitution creates an alternative acceptor splice site in intron 21, inserting two codons in the PDGFRB transcript. Functional studies revealed that the splice change induced a partial loss of function, contrasting with previously-described variants. In four tumor samples, we identified a second somatic hit at position Asp850 in PDGFRB exon 18, triggering constitutive receptor activation and resistance to imatinib. In addition to vinblastine and methotrexate, two patients had received imatinib without objective response. One of them switched to dasatinib with concomitant improvement. This splice-site PDGFRB variant favors the development of myofibroma featuring an acquired oncogenic variant in the same gene and resistance to targeted therapy.

Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia

AbstractOncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Loss of this CTCF-bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (−644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that “promoter tethering” of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

SLC12A1 variant c.1684+1 G>A causes Bartter syndrome type 1 by promoting exon 13 skipping.

Bartter syndrome type 1, an autosomal recessive genetic disorder, is caused by pathogenic loss-of-function variants in the SLC12A1 gene. It is characterized by metabolic alkalosis and prenatal-onset polyuria leading to polyhydramnios. We identified pathogenic gene in a 12-day-old newborn boy with Bartter syndrome type 1 using whole-exome sequencing. Sanger sequencing validated the identified variants. A minigene assay was performed to investigate the effect of a novel splice site variant on pre-mRNA splicing. We found a compound heterozygous variants in the SLC12A1 gene, consisting of a known pathogenic missense mutation (NM_000338: c.769 G>A; p.Gly257Ser) and a novel splice site variant (c.1684+1 G>A). In silico predictions and an in vitro minigene splicing assay demonstrated that the splicing variant c.1684+1 G>A abolished a consensus splice donor site of SLC12A1 intron 13, resulting in complete exon 13 skipping, translational frameshift, and premature termination codon, ultimately leading to loss of SLC12A1 function. Using a cell-based in vitro assay, we revealed the aberrant effect of the pathogenic splicing variant SLC12A1 c.1684+1 G>A on pre-mRNA splicing. Our findings expand the gene mutation spectrum of Bartter syndrome type 1, providing a basis for genetic diagnosis and the development of genetic medicines.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

KHSRP ameliorates acute liver failure by regulating pre-mRNA splicing through its interaction with SF3B1

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.

Single-Base Substitution Causing Dual-Exon Skipping Event in PKD2 Gene: Unusual Molecular Finding from Exome Sequencing in a Patient with Autosomal Dominant Polycystic Kidney Disease.

Background: Pathogenic variants in the Polycystic Kidney Disease 2 (PKD2) gene are associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD) in approximately 30% of cases. In recent years, the high-throughput sequencing techniques have significantly increased the number of variants identified in affected patients. Here, we described the peculiar effect of a PKD2 splicing variant, the c.1717-2A>G, identified in an Italian male patient with ADPKD. This variant led to the unusual and rare skipping of two consecutive exons, causing a large in-frame deletion. Methods: The genetic evaluation of the patient was performed using the Next-Generation Sequencing (NGS) assay Clinical Exome Solution® (SOPHiA Genetics). Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). Prediction of pathogenicity was carried out by integrating several in silico tools. RNA evaluation was performed to test the effect of the variant on the PKD2 splicing using a Reverse-Transcription PCR coupled with cDNA sequencing. Results: NGS revealed the presence of the PKD2 c.1717-2A>G variant that lies in the canonical splice site of intron 7. This rare variant was predicted to have a significant impact on the splicing, proved by the RNA-based analysis. We identified the presence of a transcript characterised by the simultaneous skipping of exons 8 and 9, with a retained reading frame and the merging of exons 7-10. Conclusions: We described for the first time a dual-exon skip event related to the presence of a single-base substitution in the PKD2 gene in an ADPKD-affected patient. We assumed that the molecular basis of such a rare mechanism lies in the specific order of intron removal. The finding represents novel evidence of an alternative and unusual splicing mechanism in the PKD2 gene, adding insights to the pathogenesis of the ADPKD.

Transcriptomic analysis reveals regulation of adipogenesis via long non-coding RNA, alternative splicing, and alternative polyadenylation

Obesity is characterized by dysregulated adipogenesis that leads to increased number and/or size of adipocytes. Understanding the molecular mechanisms governing adipogenesis is therefore key to designing therapeutic interventions against obesity. In our study, we analyzed 3’-end sequencing data that we generated from human preadipocytes and adipocytes, as well as previously published RNA-seq datasets, to elucidate mechanisms of regulation via long non-coding RNA (lncRNA), alternative splicing (AS) and alternative polyadenylation (APA). We discovered lncRNAs that have not been previously characterized but may be key regulators of white adipogenesis. We also detected 100 AS events and, using motif enrichment analysis, identified RNA binding proteins (RBPs) that could mediate exon skipping—the most prevalent AS event. In addition, we show that usage of alternative poly(A) sites in introns or 3’-UTRs of key adipogenesis genes leads to isoform diversity, which can have significant biological consequences on differentiation efficiency. We also identified RBPs that may modulate APA and defined how 3’-UTR APA can regulate gene expression through gain or loss of specific microRNA binding sites. Taken together, our bioinformatics-based analysis reveals potential therapeutic avenues for obesity through manipulation of lncRNA levels and the profile of mRNA isoforms via alternative splicing and polyadenylation.

A novel splice-altering TNC variant (c.5247A > T, p.Gly1749Gly) in an Chinese family with autosomal dominant non-syndromic hearing loss

BackgroundThis study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene.MethodsA five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis.ResultsThe family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon–intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19.ConclusionThis novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

Multiplexed single-cell characterization of alternative polyadenylation regulators

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.

Identification and analysis of nine new flo2 allelic mutants in rice

FLO2 is involved in grain development and storage substance synthesis in rice, and therefore can regulate grain size and quality. In this study, we identified 4 new flo2 allelic mutants with nonsense and frameshift mutation in the exon of 6, 10, 11 and 21 and 5 new flo2 allelic mutants with alternative splicing and frameshift mutation at the splicing site of intron 13, 14, 16 and 17. Compared with wild-type rice, the outer endosperm of flo2 mutants was transparent, and the inner endosperm was floury. Different mutation sites and types of FLO2 significantly decreased kernel width, thickness and weight to some extent. The contents of storage protein, starch, amylose and amylopectin showed significant decrease at different levels among 9 flo2 mutants. The expressions of most storage protein synthesis genes and starch synthesis-related genes were significantly down-regulated, and exhibited different ranges of variation among different flo2 mutants. This study could add helpful information for the roles of flo2 alleles in rice quality regulation and provide abundant germplasm resources for rice quality breeding.

Generation of an induced pluripotent stem cell line, JHUi005-A, from a Marfan Syndrome patient harboring a pathogenic c.3338-2A>C intronic splicing variant

Marfan Syndrome, a connective tissue disorder caused by Fibrillin-1 (FBN1) gene mutations, induces disease in the ocular, musculoskeletal, and cardiovascular systems and increases aortic vulnerability to rupture associated with high mortality rates. We describe an induced pluripotent stem cell line (HFD1) generated from patient-derived human dermal fibroblasts harboring a heterozygous c.3338-2A>C intronic splice acceptor site variant preceding Exon 28 of FBN1. The clonal line, which produces abnormal FBN1 splice variants, has a normal karyotype, expresses appropriate stemness markers, and maintains trilineage differentiation potential. This line represents a valuable resource for studying how abnormal splicing variants contribute to Marfan Syndrome.

Abstract PR003: Mechanisms of therapeutic vulnerability of mismatch repair defective cancers to RNA polymerase I inhibitors

Abstract The goal of this study was to identify cancer types with synthetic vulnerability to RNA polymerase I (Pol I) inhibitors and to resolve mechanistic underpinnings of this vulnerability. Specific and selective Pol I inhibitors, pioneered by our group, block the transcription of rRNA, which is the rate-limiting step in ribosome biogenesis and protein synthesis. We conducted unbiased large-scale cancer cell line screens with the Pol I inhibitors and performed correlative analyses using DepMap and Achilles databases to identify cancer types and features that sensitize cancer cells to these inhibitors. The most sensitive lineages were colorectal, endometrial, esophageal, leukemia, myeloma, and ovarian. Microsatellite instable (MSI) cancer cell lines were highly sensitive and significantly more sensitive than microsatellite stable lines. Comparison of the cancer cell line responses to the Achilles gene essentiality database showed high correlation of the sensitivity to genetic knock-out of three Pol I subunits. This unbiased finding strongly supports identification of Pol I as the target. Correlative analyses for vulnerability identified two ribosomal proteins, RPL22 and RPL22L1, and MDM4, a p53 transcriptional repressor as the top candidates. Notably, RPL22K15Rfs hotspot mutation, the top-ranking genetic correlate, is present in up 70% MSI cancer cell lines and tumors. RPL22 and RPL22L1 are a synthetic lethal paralog pair. RPL22 is a negative regulator of RPL22L1 and consequently, haploinsufficiency or loss of RPL22 induces RPL22L1. RPL22L1 was the top-ranking expression correlate. Lastly, MDM4 was the top protein marker and is highly expressed in MSI cancers. These findings were validated using genetic and mechanistic analyses. We show that either genetic or chemical inhibition of the Pol I enzyme decreases the expression of RPL22L1 and MDM4 and alters their splicing. RPL22L1 is spliced to an alternative 3’ splice site in intron 2 leading to an early truncation. MDM4 exon 6 skipping converts the full-length p53 repressive form to a short NMD-targeted form. Mechanistically, by using GoldCLIP-seq, rMATS and splicing analyses, we show that RPL22 is a master regulator of splicing by binding to intronic sequences and splice sites of RPL22L1 and MDM4. Finally, we show the efficacy and tolerance of the Pol I inhibitors in xenograft and patient-derived MSI models expressing these markers. In summary, this synthetic vulnerability screen using new Pol I inhibitors uncovered a connection between Pol I transcription and cellular splicing programs and identified perturbation of this connection in MSI cancers. We conclude that RPL22 mutation could serve as tumor-agnostic biomarker for targeting cancers by Pol I inhibitors. Furthermore, this work identifies a new Pol I transcription-dependent ribosomal protein-governed pathway that converges on the regulation of p53. Citation Format: Marikki Laiho, Wenjun Fan, Hester Liu, Gregory C. Stachelek, Asma Begum, Catherine E. Davis, Tony Dorado, Pablo de Leon, Rajeshkumar N.V., James C. Barrow. Mechanisms of therapeutic vulnerability of mismatch repair defective cancers to RNA polymerase I inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR003.

A rare ACAN non-canonical splicing-site intron variant results in familial short stature

ObjectiveACAN gene variants, prevalent monogenic defects linked to short stature, are characterized by impaired cartilage generation in growth plates. We aimed to unravel the genetic basis of short stature in a specific pedigree by investigating the role of a novel non-canonical splicing-site variant, c.630-13G > A, within the ACAN gene. MethodSanger sequencing was used for pedigree verification, and the effects of this variant on mRNA splicing were analyzed through minigene assay. ResultsThe study revealed that this variant led to the creation of a previously unreported splice site in the fourth intron, resulting in the incorporation of an 11 bp sequence from the intron into the final transcript. This alteration led to a frameshift and formation of a premature termination codon, impacting the structure of the aggrecan protein. ConclusionsWe document the pathogenicity of an ACAN non-canonical splicing-site variant, emphasizing the significance of considering intronic variants during genetic testing.

Lineage-specific splicing regulation of MAPT gene in the primate brain

Divergence of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is widespread in mammals, including primates, but the underlying mechanisms and functional impact are poorly understood. Here, we modeled cassette exon inclusion in primate brains as a quantitative trait and identified 1,170 (∼3%) exons with lineage-specific splicing shifts under stabilizing selection. Among them, microtubule-associated protein tau (MAPT) exons 2 and 10 underwent anticorrelated, two-step evolutionary shifts in the catarrhine and hominoid lineages, leading to their present inclusion levels in humans. The developmental-stage-specific divergence of exon 10 splicing, whose dysregulation can cause frontotemporal lobar degeneration (FTLD), is mediated by divergent distal intronic MBNL-binding sites. Competitive binding of these sites by CRISPR-dCas13d/gRNAs effectively reduces exon 10 inclusion, potentially providing a therapeutically compatible approach to modulate tau isoform expression. Our data suggest adaptation of MAPT function and, more generally, a role for AS in the evolutionary expansion of the primate brain.

Identification of a Novel Intronic Mutation in VMA21 Associated with a Classical Form of X-Linked Myopathy with Autophagy.

Introduction VMA21 -related myopathy is one of the rare forms of slowly progressive myopathy observed in males. Till now, there have been only a handful of reports, mainly from Europe and America, and two reports from India. Method Here, we describe a case of genetically confirmed VMA21 -associated myopathy with clinical, histopathological, and imaging features with a list of known VMA21 mutations. Results A 29-year-old man had the onset of symptoms at 18 years of age with features of proximal lower limb weakness. Muscle magnetic resonance imaging showed the preferential involvement of vasti and adductor magnus. A biopsy of the left quadriceps femoris showed features of autophagic vacuolar myopathy with vacuoles containing granular eosinophilic materials. In targeted next-generation sequencing, hemizygous mutation in the 3' splice site of intron 2 of the VMA21 gene (c.164-7 T > A) was identified and confirmed the diagnosis of X-linked myopathy with excessive autophagy. Conclusion This report expands the phenotypic and genotypic profile of VMA21 -related myopathy, with a yet unreported mutation in India.

Abstract 2063: Endothelial Cell Scavenger Receptor Class B, Type I Expression Is Transcriptionally Controlled By HIF-1α

Background: Scavenger Receptor Class B, Type I (SR-BI), encoded by Scarb1, mediates LDL cholesterol transcytosis in endothelial cells (EC) to deliver circulating LDL into the subendothelial space to drive atherogenesis. Research Questions: It is unknown whether EC SR-BI expression is altered in atherosclerosis, and how EC SR-BI expression is regulated. Approach: Single cell RNAseq was performed on coronary arteries from 5 ischemic heart disease patients, in segments both with and without a grossly apparent atheroma. Transcription factor (TF) network analysis was done to reveal possible regulatory relationships between differentially-expressed genes (DEGs). The modulation of EC SR-BI was studied in primary human aortic EC (HAEC) and in vivo in mouse aortic EC. ChIP-seq, ChIP-qPCR and CRISPR-based mutagenesis were used to interrogate TF binding to the human Scarb1 gene. EC SR-BI expression was determined by immunoblotting and qPCR, and EC LDL transcytosis was studied in transwells using DiI-labeled LDL. Results: SR-BI expression was greater in EC in coronary segments with atheroma versus those without atheroma (p=0.003). TF networks for DEGs in atheroma versus non-atheroma EC, and for DEGs in EC with low versus high SR-BI revealed multiple TF with possible regulatory relationships with Scarb1. One TF in common was HIF-1α. In HAEC, an increase in HIF-1α with the HIF prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) caused upregulation of SR-BI mRNA and protein, and a parallel increase in LDL transcytosis that was entirely SR-BI-dependent. Expression of a constitutively-active HIF-1α variant also increased SR-BI and LDL transcytosis. ChIP-seq revealed a HIF-1α binding site in Scarb1 intron 1, and HIF-1α binding there increased 4-fold with DMOG. CRISPR deletion of the binding site fully prevented SR-BI upregulation by DMOG without affecting known HIF-1α target genes. In mice, 3d treatment with DMOG increased SR-BI in aortic EC. Conclusions: EC expression of SR-BI is upregulated with increasing atherosclerosis severity in human coronary artery. HIF-1α is upregulated in parallel, and HIF-1α causes the direct transcriptional upregulation of EC SR-BI leading to enhanced EC LDL transport. HIF-1α control of SR-BI in EC may be critical to atherogenesis.

A novel homozygous splice site variant in ARL2BP causes a syndromic autosomal recessive rod-cone dystrophy with situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts

BackgroundThis report presents a clinical case of syndromic rod-cone dystrophy due to a splice site variant in the ARL2BP gene causing situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts. The presence of renal agenesis and cryptorchidism expands the clinical manifestations due to ARL2BP variants. The detailed, long-term follow-up contributes valuable insights into disease progression, aiding clinical diagnosis and patient management.Case PresentationThe male patient complained of photophobia as the first symptom when he was 20 years old followed by nyctalopia, loss of central visual acuity and peripheral visual field ten years later. Genetic analysis identified a likely pathogenic homozygous variant (c.294-1G > C) involving the splicing acceptor site of intron 4. Reported symptoms together with full-field stimulus threshold testing, electroretinogram and advanced multimodal imaging allowed us to recognize the typical characteristics of a mixed retinal dystrophy. Despite the end-stage retinal disease, this patient still retained a useful residual vision at 63 years and had a slow disease progression during the last 5 years of evaluation.Discussion and conclusionsOur findings underscore the variable clinical presentation of ARL2BP variants, emphasizing the importance of a nuanced approach in diagnosing and managing patients. The presence of renal cysts warrants consideration of a differential diagnosis, particularly with Senior-Loken (SLS), Bardet-Biedl (BBS) and Joubert syndromes (JS) but also with Short Rib Thoracic Dysplasia 9, highlighting the need for careful phenotypic evaluation in these cases.

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes invivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.