Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that Ovol2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) driving gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and consequently induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation.

Multi-ancestry fine mapping implicates OAS1 splicing in risk of severe COVID-19

The OAS1/2/3 cluster has been identified as a risk locus for severe COVID-19 among individuals of European ancestry, with a protective haplotype of approximately 75 kilobases (kb) derived from Neanderthals in the chromosomal region 12q24.13. This haplotype contains a splice variant of OAS1, which occurs in people of African ancestry independently of gene flow from Neanderthals. Using trans-ancestry fine-mapping approaches in 20,779 hospitalized cases, we demonstrate that this splice variant is likely to be the SNP responsible for the association at this locus, thus strongly implicating OAS1 as an effector gene influencing COVID-19 severity.

Acid-Base Effects of Combined Renal Deletion of NBCe1-A and NBCe1-B

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deleting the A splice variant of the Na⁺-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex, but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The current studies determined the effects of combined renal deletion of NBCe1-A and NBCe1-B on systemic and proximal tubule ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. Since renal NBCe1-A and NBCe1-B expression is limited to the proximal tubule, Cre-positive mice had proximal tubule NBCe1-A/B deletion (PT-NBCe1-A/B KO). While on basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased expression of phosphate-dependent glutaminase (PDG) and phospho­enol­pyruvate carboxy­kinase (PEPCK) and increased expression of glutamine synthetase (GS), an ammonia recycling enzyme, in PT in both the cortex and OM. Exogenous acid-loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in PDG, PEPCK, and GS expression in the proximal tubule in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to proximal tubule ammonia metabolism in the OM and thereby to systemic acid-base regulation.

Oncogenic role of a developmentally regulated NTRK2 splice variant

Temporally-regulated alternative splicing choices are vital for proper development yet the wrong splice choice may be detrimental. Here we highlight a novel role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in both humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis and forced over-expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring TRK fusions. Affinity purification-mass spectrometry (AP-MS) proteomic analysis reveals TrkB.T1 has distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, TGF-β, Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

Cistrome And Transcriptome Analysis Identifies Unique Androgen Receptor (AR) And AR- V7 Splice Variant Chromatin Binding And Transcriptional Activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR- V7 splice variant chromatin binding and transcriptional activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication

HBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

Retroperitoneal leiomyosarcoma in a female patient with a germline splicing variant RAD51D c.904-2A > T: a case report

Background RAD51D (RAD51 paralog D) is an intermediate cancer susceptibility gene for primary ovarian cancer, including fallopian tube and peritoneal carcinomas and breast cancer. Although gynecological non-epithelial tumors such as uterine sarcomas are associated with genomic instability, including BRCA impairment, there is no clear evidence of the relationship between RAD51D variants and the risk of sarcoma development. Case presentation A Japanese woman in her 50s underwent multiple surgical resections and several regimens of chemotherapy for tumors that originated in the retroperitoneum and recurred in the peritoneum over a clinical course of approximately 4 years. The peritoneal tumor was histologically diagnosed as a leiomyosarcoma and was genetically identified to show a splice variant of RAD51D c.904-2A > T [NM_002878] through tumor profiling performed as a part of cancer precision medicine. The confirmatory genetic test performed after genetic counseling revealed that the RAD51D splicing variant detected in her tumor was of germline origin. In silico analyses supported the possible pathogenicity of the detected splice variant of RAD51D with a predicted attenuation in mRNA transcription and truncated protein production due to frameshifting, which was attributed to a single-nucleotide alteration in the splicing acceptor site at the 3′-end of intron 9 of RAD51D. Considering her unfavorable clinical outcome, which showed a highly aggressive phenotype of leiomyosarcoma with altered RAD51D, this case provided novel evidence for the relationship of a RAD51D splicing variant with malignant tumor development or progression. We report the findings of this rare case with possible involvement of the germline variant of RAD51D c.904-2A > T as a potential predisposing factor for malignant tumors, including leiomyosarcoma. Conclusions We present the findings of a case of leiomyosarcoma in the peritoneum of a female patient with a novel germline splicing variant of RAD51D as potential evidence for the pathogenicity of the variant and its involvement in the risk of sarcoma etiology and/or development. To the best of our knowledge, this is the first case report describing a leiomyosarcoma carrying a germline RAD51D splicing variant and elucidating its pathogenicity on the basis of computational prediction of the impairment of normal transcription and the presumed loss of functional protein production.