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The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC. Dysregulation of mRNA alternative splicing is prevalent in cancers. Here, the authors characterized the landscape of aberrant alternative splicing during the development of prostate cancer, progression and therapeutic resistance and show that splicing modulator, E7107, reduces growth in castration-resistant prostate cancer.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7 , we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Antisense oligonucleotides effectively decrease the inclusion of exon  8A of CACNA1C in human cells both in vitro and in rodents transplanted with human brain organoids, and a single intrathecal administration rescued both calcium changes and in vivo dendrite morphology of patient neurons.

Key Points All RNA-targeted therapeutic technologies exploit oligonucleotides that bind to target RNA, but they differ in their mechanism of action and produce different effects. Small interfering RNAs, antisense oligonucleotides and external guide sequences lead to enzyme-dependent degradation of targeted mRNA. Drugs involving these approaches are designed to reduce the level of harmful gene products such as viral or bacterial proteins or disease-promoting cellular proteins. They could be useful against cancer as well as viral and bacterial infections, or used to prevent the accumulation of high levels of cholesterol in the bloodstream. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading RNA. Splice-switching oligonucleotides are discussed in detail in this Review; these oligonucleotides redirect alternative splicing, repair defective RNA or restore the production of proteins that are missing because of genetic defects. Splice-switching oligonucleotide-based drugs should be useful for the treatment of genetic diseases such as Duchenne muscular dystrophy, spinal muscular atrophy and β-thalassaemia. Compared to classical small-molecule drugs, it is much more difficult to achieve intracellular delivery with oligonucleotides; this is still a major issue for this class of drugs. The advantage of oligonucleotides is their high specificity, which results from sequence-specific base pairing to target RNA. The oligonucleotide-based drug fomivirsen was approved by the US Food and Drug Administration in 1998 for the treatment of viral retinitis in patients with AIDS. Oligonucleotide-based drugs are now in advanced clinical trials for the treatment of cancer and Duchenne muscular dystrophy as well as for lowering high cholesterol levels. Here, the authors highlight how RNA-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression, and so may be useful for treating disorders such as Duchenne muscular dystrophy, spinal muscular atrophy and β-thalassaemia. Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degradation of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression. Moreover, they can be extensively chemically modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to achieving its clinical potential.

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells. The cytokine IFNα is expressed in the islets of individuals with type 1 diabetes and contributes to local inflammation and destruction of beta cells. Here, the authors provide a global multiomics view of IFNα-induced changes in human beta cells at the level of chromatin, mRNA and protein expression.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

The ability to control gene expression in mammalian cells is crucial for safe and efficacious gene therapies and for elucidating gene functions. Current gene regulation systems have limitations such as harmful immune responses or low efficiency. We describe the pA regulator, an RNA-based switch that controls mammalian gene expression through modulation of a synthetic polyA signal (PAS) cleavage introduced into the 5′ UTR of a transgene. The cleavage is modulated by a ‘dual-mechanism’—(1) aptamer clamping to inhibit PAS cleavage and (2) drug-induced alternative splicing that removes the PAS, both activated by drug binding. This RNA-based methodology circumvents the immune responses observed in other systems and achieves a 900-fold induction with an EC 50 of 0.5 µg ml − 1 tetracycline (Tc), which is well within the FDA-approved dose range. The pA regulator effectively controls the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability. Mammalian gene expression is controlled by an RNA switch system.

Background Dysregulation of alternative splicing plays a pivotal role in tumorigenesis and metastasis in triple-negative breast cancer (TNBC). Serine/arginine-rich (SR) proteins, essential components of the spliceosome, undergo phosphorylation by Cdc2-like kinase (CLK). Here we explored the impact of pharmacological inhibition of CLK using a novel inhibitor, T-025, on the spliceosome complex and transcriptional responses in relation to cell proliferation and migration in TNBC. Methods We evaluated the anti-proliferative and anti-migratory efficacy of T-025 in a spectrum of TNBC cell lines. Fluorescent reporter cell lines and flowcytometry were used to determine the effect of T-025 on cell cycle. Deep RNA sequencing was performed to unravel the differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) upon T-025 treatment. Pulldown/MS was used to uncover the impact of T-025 on SRSF7 interactome. Live-cell imaging and photobleaching experiments were conducted to determine the subnuclear localization of SRSF7-GFP and its dynamic mobility. Results T-025 exhibited a potent anti-proliferative effect in a spectrum of TNBC cell lines, particularly in highly proliferative cell lines. Treatment with T-025 induced cell cycle arrest in the G1-S phase, resulting in an increased proportion of aneuploidy cells and cells with 4 N DNA. T-025 significantly inhibited cell migration in highly migratory TNBC cell lines. Deep RNA sequencing uncovered numerous DEGs and ASGs upon T-025 treatment, which were significantly enriched in pathways related to cell division, RNA splicing and cell migration. Pulldown/MS showed that SRSF7 interacted more with nuclear-speckle-residing proteins, while less with RNA helicases and polymerases upon T-025 treatment. Enhanced interactions between SRSF7 and other phosphorylated SR proteins localized at nuclear speckles were also observed. Live-cell imaging indicated that T-025 treatment induced the accumulation of SRSF7-GFP at nuclear speckles and nuclear speckles’ enlargement, restricting its protein dynamic mobility. Conclusions CLK inhibition using T-025 leads to the accumulation of splicing factors at nuclear speckles and stalls their release to splicing sites, resulting in the RNA splicing reprogramming of a large number of genes involved in cell division, migration and RNA splicing. Our findings provide evidence that T-025 could be a promising therapeutic drug for TNBC patients.

So far, gene therapies have relied on complex constructs that cannot be finely controlled 1 , 2 . Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote X on , does not require the co-expression of any regulatory proteins. Using X on , the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of X on to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the X on switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans. A switch system is developed to control the expression of therapeutic genes, involving the administration of a small-molecule drug to induce splicing-mediated control of mRNA translation.

Background Alternative splicing (AS) is consistently linked to tumor progression. SRSF1, the first identified proto-oncogene in the serine/arginine-rich splicing factor (SRSF) protein family, plays a crucial role. However, the specific functions and potential mechanisms of SRSF1 in advancing bladder cancer (BCa) progression and influencing chemosensitivity remain largely unexplored. Methods The expression of SRSF1 in BCa tissues and cell lines was investigated using quantitative real-time PCR (RT-qPCR) and western blotting. Survival analysis was employed to examine the association between SRSF1 expression and prognosis of BCa. The functions of SRSF1 were evaluated through proliferation assays, migration assays, IC50 determination assays, and tumorigenesis assays in nude mice. Subsequent RNA sequencing validated the relationship between SRSF1 alternative splicing and the mitophagy pathway. Mitochondrial membrane potential (MMP) was assessed using JC-1 staining. Mitophagy and autophagic flux were quantified using transmission electron microscopy and fluorescence imaging. RNA immunoprecipitation, CUT & RUN assays, and luciferase reporter assays were performed to validate the SRSF1/HIF1A/BNIP3 axis. Results High expression of SRSF1 in BCa was significantly associated with poor prognosis. SRSF1 promoted the progression of BCa cells and conferred resistance to cisplatin both in vitro and in vivo. Mechanistically, SRSF1 interacted with pre-HIF1A via the RRM1/RRM2 domain, thereby enhancing the production of the transcription factor HIF1A through the alternative splicing pathway. This interaction subsequently activated the HIF1A/BNIP3 axis, which promoted mitophagy in BCa. Ultimately, this led to further progression of bladder cancer and a decrease in cisplatin sensitivity. Conclusions SRSF1 indicated poor prognosis and promoted the progression and cisplatin resistance of BCa cells through the HIF1A/BNIP3/mitophagy axis. It holds significant potential as a novel biomarker for the diagnosis and treatment of BCa, particularly in chemotherapy.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP- familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP- FD exon 20 has a suboptimal 5′ splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases. Familial dysautonomia is caused by splicing mutation of IKBKAP gene, which induces skipping of exon 20 and subsequent functional loss. Here, the authors report that a synthetic splice modulator RECTAS ameliorates pathogenic exon 20 skipping and shows therapeutic effects in cellular and animal models.