Expression Of Splice Isoforms Research Articles

SARS-CoV-2 Selectively Induces the Expression of Unproductive Splicing Isoforms of Interferon, Class I MHC, and Splicing Machinery Genes

RNA processing is a highly conserved mechanism that serves as a pivotal regulator of gene expression. Alternative processing generates transcripts that can still be translated but lead to potentially nonfunctional proteins. A plethora of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategically manipulate the host’s RNA processing machinery to circumvent antiviral responses. We integrated publicly available omics datasets to systematically analyze isoform-level expression and delineate the nascent peptide landscape of SARS-CoV-2-infected human cells. Our findings explore a suggested but uncharacterized mechanism, whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling, interferon-stimulated (ISGs), class I MHC, and splicing machinery genes, including IRF7, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6 and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs an unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.

Cancer and Autism: How PTEN Mutations Degrade Function at the Membrane and Isoform Expression in the Human Brain

Mutations causing loss of PTEN lipid phosphatase activity can promote cancer, benign tumors (PHTS), and neurodevelopmental disorders (NDDs). Exactly how they preferentially trigger distinct phenotypic outcomes has been puzzling. Here, we demonstrate that PTEN mutations differentially allosterically bias P loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations of the functional wild-type state, while sampled conformations for the strong, cancer-related driver mutation hotspots favor catalysis-primed conformations, suggesting that NDD mutations are likely to be weaker, and our large-scale simulations show why. Prenatal PTEN isoform expression data suggest exons 5 and 7, which harbor NDD mutations, as cancer-risk carriers. Since cancer requires more than a single mutation, our conformational and genomic analysis helps discover how same protein mutations can foster different clinical manifestations, articulates a role for co-occurring background latent driver mutations, and uncovers relationships of splicing isoform expression to life expectancy.

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11β splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11βki/− mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11βki/− males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11β and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

Abstract 3845: Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain

Abstract PTEN negatively regulates PI3K/AKT/mTOR signaling through dephosphorylation of PIP3 to PIP2. PTEN dysfunction triggers disease phenotypes, ranging from cancer and PHTS to NDDs such as ASD. The most common PTEN mutations, nonsense, frameshift, and deletion/insertion, may cause premature termination of translation. PTEN has a considerable number of missense mutations that may result in loss of protein function through reduced catalytic activity and protein stability. To decipher mechanistic details of the mutations and structural features of the mutant proteins, comprehensive computational studies were performed for PTEN mutants at an anionic lipid bilayer containing PC, PS, PIP2, and PIP3. Six PTP mutations (Y68H, H93R, A126T, R130Q, G132D, R173C) and two C2 mutations (F241S, D252G) were considered. The PTP mutations are known to associate with cancer, PHTS, macrocephaly, and ASD, while the C2 mutations are exclusively related to macrocephaly and ASD. Our studies indicate that the PTEN mutants can effectively absorb the anionic lipid bilayer, similar to wild-type PTEN. However, Y68H significantly disrupts the core of PTP domain, reducing protein stability. H93R hijacks the substate PIP3, interrupting the catalytic site residues recruitment of the substate for catalysis. A126T, R130Q, and G132D in the P loop directly influence the dynamics of the loop, yielding a collapsed loop conformation. The collapsed P loop loses the interaction with the WPD loop, leading to dehydration in the catalytic site. R173C and D252G at the interface between two major domains disrupt the domain-domain interaction, allosterically biasing the P loop dynamics. However, F241S in C2 exhibits less effective allosteric connection to the catalytic site than that observed in the wild type. PCA of the sampled conformations found that the macrocephaly and ASD-related H93R and F241S are likely to sample the conformations present in the wild type. In contrast, the sampled conformations for the cancer and PHTS-related Y68H, A126T, and G132D are different from those for the wild type. These distinct structural features seem to correlate with mutation strength and timing of the expression of the genes that determine the cancer and NDD outcomes. The isoform expression data extracted from the BrainSpan indicate that exon 5 is impacted by NDD or non-NDD mutations. Interestingly, PTEN splicing isoforms that do not carry exon 5 are exclusively impacted by the NDD mutations, F241S and D252G. The increased life expectancy of PTEN variants carriers carrying exons 5 and 7 is highly correlated to increased lifetime risk for certain cancers. Our results underscore the detailed structural and functional mechanisms of PTEN with mutations at the membrane, and suggest that tissue-specific splicing isoform expression should be taken into consideration while predicting the effect of mutations in various diseases. Citation Format: Hyunbum Jang, Jiaye Chen, Lilia M. Iakoucheva, Ruth Nussinov. Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3845.

Malignant DFFB isoform switching promotes leukemia survival in relapse pediatric T-cell acute lymphoblastic leukemia.

With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T-cell phenotype (T-ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T-ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA-sequencing analysis on patient-derived samples followed by splice isoform-specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T-ALL. Moreover, patients with T-ALL displayed pro-survival splice isoform switching favoring pro-survival isoforms compared to normal healthy stem cells. Cumulatively, pro-survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T-ALL proliferation and survival, thus serving as a potential therapeutic option.

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS. Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays. We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140. We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (EFCAB13), rs1131123 (HLA-C), rs10783847 (TSFM), and rs2014886 (TSFM) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed. In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

Identification of Candidate mRNA Isoforms for Prostate Cancer-Risk SNPs Utilizing Iso-eQTL and sQTL Methods.

Single nucleotide polymorphisms (SNPs) impacting the alternative splicing (AS) process (sQTLs) or isoform expression (iso-eQTL) are implicated as important cancer regulatory elements. To find the sQTL and iso-eQTL, we retrieved prostate cancer (PrCa) tissue RNA-seq and genotype data originating from 385 PrCa European patients from The Cancer Genome Atlas. We conducted RNA-seq analysis with isoform-based and splice event-based approaches. The MatrixEQTL was used to identify PrCa-associated sQTLs and iso-eQTLs. The overlap between sQTL and iso-eQTL with GWAS loci and those that are differentially expressed between cancer and normal tissue were identified. The cis-acting associations (FDR < 0.05) for PrCa-risk SNPs identified 42, 123, and 90 PrCa-associated cassette exons, intron retention, and mRNA isoforms belonging to 25, 95, and 83 genes, respectively; while assessment of trans-acting association (FDR < 0.05) yielded 59, 65, and 196 PrCa-associated cassette exons, intron retention and mRNA isoforms belonging to 35, 55, and 181 genes, respectively. The results suggest that functional PrCa-associated SNPs can play a role in PrCa genesis by making an important contribution to the dysregulation of AS and, consequently, impacting the expression of the mRNA isoforms.

Detailed Analysis of Dorsal-Ventral Gradients of Gene Expression in the Hippocampus of Adult Rats.

We performed RNA sequencing of the dorsal and ventral parts of the hippocampus and compared it with previously published data to determine the differences in the dorsoventral gradients of gene expression that may result from biological or technical variability. Our data suggest that the dorsal and ventral parts of the hippocampus differ in the expression of genes related to signaling pathways mediated by classical neurotransmitters (glutamate, GABA, monoamines, etc.) as well as peptide and Wnt ligands. These hippocampal parts also diverge in the expression of axon-guiding molecules (both receptors and ligands) and splice isoforms of genes associated with intercellular signaling and cell adhesion. Furthermore, analysis of differential expressions of genes specific for astrocytes, microglia, oligodendrocytes, and vascular cells suggests that non-neuronal cells may also differ in the characteristics between hippocampal parts. Analysis of expression of transposable elements showed that depletion of ribosomal RNA strongly increased the representation of transposable elements in the RNA libraries and helped to detect a weak predominance of expression of these elements in the ventral hippocampus. Our data revealed new molecular dimensions of functional differences between the dorsal and ventral hippocampus and points to possible cascades that may be involved in the longitudinal organization of the hippocampus.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a β-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of β-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 (Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression.

Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles.

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.

Pan-Cancer Analysis Reveals Alternative Splicing Characteristics Associated With Immune-Related Adverse Events Elicited by Checkpoint Immunotherapy.

Immune-related adverse events (irAEs) can impair the effectiveness and safety of immune checkpoint inhibitors (ICIs) and restrict the clinical applications of ICIs in oncology. The predictive biomarkers of irAE are urgently required for early diagnosis and subsequent management. The exact mechanism underlying irAEs remains to be fully elucidated, and the availability of predictive biomarkers is limited. Herein, we performed data mining by combining pharmacovigilance data and pan-cancer transcriptomic information to illustrate the relationships between alternative splicing characteristics and irAE risk of ICIs. Four distinct classes of splicing characteristics considered were associated with splicing factors, neoantigens, splicing isoforms, and splicing levels. Correlation analysis confirmed that expression levels of splicing factors were predictive of irAE risk. Adding DHX16 expression to the bivariate PD-L1 protein expression-fPD1 model markedly enhanced the prediction for irAE. Furthermore, we identified 668 and 1,131 potential predictors based on the correlation of the incidence of irAEs with splicing frequency and isoform expression, respectively. The functional analysis revealed that alternative splicing might contribute to irAE pathogenesis via coordinating innate and adaptive immunity. Remarkably, autoimmune-related genes and autoantigens were preferentially over-represented in these predictors for irAE, suggesting a close link between autoimmunity and irAE occurrence. In addition, we established a trivariate model composed of CDC42EP3-206, TMEM138-211, and IRX3-202, that could better predict the risk of irAE across various cancer types, indicating a potential application as promising biomarkers for irAE. Our study not only highlights the clinical relevance of alternative splicing for irAE development during checkpoint immunotherapy but also sheds new light on the mechanisms underlying irAEs.

ADAR1 Splicing Modulation As a Mechanism to Eradicate Immunologically Silent Leukemia Stem Cells

Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. DisclosuresCrews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Full-length isoform transcriptome of the developing human brain provides further insights into autism.

SUMMARYAlternative splicing plays an important role in brain development, but its global contribution to human neurodevelopmental diseases (NDDs) requires further investigation. Here we examine the relationships between splicing isoform expression in the brain and de novo loss-of-function mutations from individuals with NDDs. We analyze the full-length isoform transcriptome of the developing human brain and observe differentially expressed isoforms and isoform co-expression modules undetectable by gene-level analyses. These isoforms are enriched in loss-of-function mutations and microexons, are co-expressed with a unique set of partners, and have higher prenatal expression. We experimentally test the effect of splice-site mutations and demonstrate exon skipping in five NDD risk genes, including SCN2A, DYRK1A, and BTRC. Our results suggest that the splice site mutation in BTRC reduces translational efficiency, likely affecting Wnt signaling through impaired degradation of β-catenin. We propose that functional effects of mutations should be investigated at the isoform- rather than gene-level resolution.

Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene.

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

CLK inhibition alters tau splicing and has a neuroprotective effect in vitro

AbstractBackgroundMicrotubule‐associated protein tau (MAPT) undergoes alternative splicing at exon 10 which encodes one of its four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau isoforms with all four repeats (4R) whilst exon 10 exclusion leads to isoforms with three repeats (3R). The 1:1 ratio between 3R and 4R isoforms is tightly regulated by splicing machinery and crucial to normal function. Kinases such as CLKs regulate the activity of splicing factors and their interaction with target transcripts, including MAPT. Disruption to this balance leads to the formation of neurofibrillary tangles (NFTs) and neurodegeneration but the mechanism of this is not yet wholly understood.MethodsDifferentiated Neuro2a and SH‐SY5Y cells were used as neuronal models as these cell lines show an imbalance of MAPT isoforms with bias towards 4R and 3R respectively. Splicing factor SRSF1 was transiently knocked down to confirm its role in MAPT splicing. Novel kinase inhibitors were used to reduce the activity of CLKs and determine the effect on MAPT splicing. The expression ratio of 4R and 3R isoforms was measured by RT‐PCR. Okadaic acid (OA) was used to produce a neurotoxic model and investigate the use of kinase inhibitors for neuronal protection. As a functional measure β3‐tubulin fluorescent marker was used to quantify OA dependent reduction in neurite outgrowth.Results4R:3R tau expression significantly decreased by 65% in Neuro2a cells with knockdown of SRSF1 (students t‐test, p&lt;0.001). CLK inhibition increased 4R:3R by 50% in SH‐SY5Y cells compared to control (one‐way ANOVA, p&lt;0.05). SHSY5Y cells neurite outgrowth was significantly reduced by 60% and 85% with 1nM and 5nM OA treatment respectively, demonstrating a concentration dependent effect (one‐way ANOVA, p&lt;0.001). At 1nM OA, neurite outgrowth was rescued by co‐treatment with a novel CLK inhibitor (two‐way ANOVA, p&lt;0.0001).ConclusionsNovel CLK inhibitors can modulate the expression of MAPT splicing isoforms and exert a functional effect on neurite outgrowth. This suggests these compounds could have a therapeutic effect by rectifying the balance of 4R:3R tau expression.

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth.

Abstract 5726: A-to-I RNA deaminase deregulation in pre-leukemia stem cell evolution

Abstract While inflammation induced ADAR1 RNA deaminases protect the human genome from retroviral integration, deregulation promotes therapeutic resistance in many malignancies. However, the combinatorial role of these deaminases in pre-leukemia stem cell (pre-LSC) evolution to therapy resistant LSC had not been elucidated. Thus, we performed whole genome sequencing (WGS) analysis of 43 CD34+pre-leukemic myeloproliferative neoplasm (MPN) samples compared with matched saliva and non-MPN controls andwhole transcriptome sequencing (RNA-seq) analysis of 113 FACS-purified hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) from MPN, acute myeloid leukemia (AML) and healthy young and aged samples. During MPN progression, inflammation-driven ADAR1 isoform p150 upregulation corresponded with increased Adenosine-to-Inosine (A-to-I) transitions in both MPN stem and progenitor populations. We identified unique A-to-I editing events in coding regions that are associated with either normal stem progenitors or with MPN progenitors. In addition, STAT3 transcript hyper-editing leads to STAT3beta splice isoform expression. Moreover, lentiviral ADAR1p150 overexpression enhanced replating as well as beta-catenin activation, which was reversed by lentiviral shRNA ADAR1 knockdown. In summary, innate immune deaminase deregulation fuels pre-LSC evolution to LSC and may represent a vital LSC therapeutic vulnerability. Citation Format: Qingfei Jiang, Frida Holm, Jane Isquith, Adam Mark, Cayla Mason, Eduardo Reynoso, Isabella Morris, Wenxue Ma, Raymond Diep, Jessica Pham, Chanond Nasamran, Guorong Xu, Roman Sasik, Sara Brin Rosenthal, Amanda Birmingham, Leslie Crews, Gabriel Pineda, Thomas Whisenant, Kathleen Fisch, Catriona Jamieson. A-to-I RNA deaminase deregulation in pre-leukemia stem cell evolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5726.

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.

Genomic characterization reveals novel mechanisms underlying the valosin-containing protein-mediated cardiac protection against heart failure

Chronic hypertension is a key risk factor for heart failure. However, the underlying molecular mechanisms are not fully understood. Our previous studies found that the valosin-containing protein (VCP), an ATPase-associated protein, was significantly decreased in the hypertensive heart tissues. In this study, we tested the hypothesis that restoration of VCP protected the heart against pressure overload-induced heart failure. With a cardiac-specific transgenic (TG) mouse model, we showed that a moderate increase of VCP was able to attenuate chronic pressure overload-induced maladaptive cardiac hypertrophy and dysfunction. RNA sequencing and a comprehensive bioinformatic analysis further demonstrated that overexpression of VCP in the heart normalized the pressure overload-stimulated hypertrophic signals and repressed the stress-induced inflammatory response. In addition, VCP overexpression promoted cell survival by enhancing the mitochondria resistance to the oxidative stress via activating the Rictor-mediated-gene networks. VCP was also found to be involved in the regulation of the alternative splicing and differential isoform expression for some genes that are related to ATP production and protein synthesis by interacting with long no-coding RNAs and histone deacetylases, indicating a novel epigenetic regulation of VCP in integrating coding and noncoding genomic network in the stressed heart. In summary, our study demonstrated that the rescuing of a deficient VCP in the heart could prevent pressure overload-induced heart failure by rectifying cardiac hypertrophic and inflammatory signaling and enhancing the cardiac resistance to oxidative stress, which brought in novel insights into the understanding of the mechanism of VCP in protecting patients from hypertensive heart failure.