Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. 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 8A2-6. 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 developed7, 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.

Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model.

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.

RNA-Binding Proteins and Alternative Splicing Genes Are Coregulated in Human Retinal Endothelial Cells Treated with High Glucose.

To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in diabetic retinopathy (DR). We devised a comprehensive work to integrate analyses of the differentially expressed genes, including differential RBPs, and variable splicing characteristics related to DR in human retinal endothelial cells induced by low glucose and high glucose in dataset GSE117238. A total of 2320 differentially expressed genes (DEGs) were identified, including 1228 upregulated genes and 1092 downregulated genes. Further analysis screened out 232 RBP genes, and 42 AS genes overlapped DEGs. We selected high expression and consistency six RBP genes (FUS, HNRNPA2B1, CANX, EIF1, CALR, and POLR2A) for coexpression analysis. Through analysis, we found eight RASGs (MDM2, GOLGA2P7, NFE2L1, KDM4A, FAM111A, CIRBP, IDH1, and MCM7) that could be regulated by RBP. The coexpression network was conducted to further elucidate the regulatory and interaction relationship between RBPs and AS. Apoptotic progress, protein phosphorylation, and NF-kappaB cascade revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to diabetic retinopathy. Furthermore, the expression of differentially expressed RBPs was validated by qRT-PCR in mouse retinal microvascular endothelial cells and retinas from the streptozotocin mouse model. The results showed that Fus, Hnrnpa2b1, Canx, Calr, and Polr2a were remarkedly difference in high-glucose-treated retinal microvascular endothelial cells and Fus, Hnrnpa2b1, Canx, and Calr were remarkedly difference in retinas from streptozotocin-induced diabetic mice compared to control. The regulatory network between identified RBPs and RASGs suggests the presence of several signaling pathways possibly involved in the pathogenesis of DR. The verified RBPs should be further addressed by future studies investigating associations between RBPs and the downstream of AS, as they could serve as potential biomarkers and targets for DR.

Cytoprotective Activity of Polyamines Is Associated with the Alternative Splicing of RAD51A Pre-mRNA in Normal Human CD4+ T Lymphocytes.

Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression and cell proliferation as well as modulation of cell signaling. They can also decrease DNA damage and promote cell survival. In the present study, we demonstrated that polyamines have cytoprotective effects on normal human CD4+ T lymphocytes but not on cancer Jurkat or K562 cells. Pretreatment of lymphocytes with polyamines resulted in a significant reduction in cells with DNA damage induced by doxorubicin, cisplatin, or irinotecan, leading to an increase in cell survival and viability. The induction of RAD51A expression was in response to DNA damage in both cancer and normal cells. However, in normal cells, putrescin pretreatment resulted in alternative splicing of RAD51A and the switch of the predominant expression from the splice variant with the deletion of exon 4 to the full-length variant. Induction of RAD51A alternative splicing by splice-switching oligonucleotides resulted in a decrease in DNA damage and cell protection against cisplatin-induced apoptosis. The results of this study suggest that the cytoprotective activity of polyamines is associated with the alternative splicing of RAD51A pre-mRNA in normal human CD4+ T lymphocytes. The difference in the sensitivity of normal and cancer cells to polyamines may become the basis for the use of these compounds to protect normal lymphocytes during lymphoblastic chemotherapy.

Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy.

OBJECTIVES: To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. METHODS: Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. RESULTS: An altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. CONCLUSIONS: Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50µM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 µM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Therapeutic Targeting of Alternative RNA Splicing in Gastrointestinal Malignancies and Other Cancers.

Recent comprehensive genomic studies including single-cell RNA sequencing and characterization have revealed multiple processes by which protein-coding and noncoding RNA processing are dysregulated in many cancers. More specifically, the abnormal regulation of mRNA and precursor mRNA (pre-mRNA) processing, which includes the removal of introns by splicing, is frequently altered in tumors, producing multiple different isoforms and diversifying protein expression. These alterations in RNA processing result in numerous cancer-specific mRNAs and pathogenically spliced events that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumor suppressor genes. Abnormally spliced pre-mRNAs are also associated with resistance to cancer treatment, and certain cancers are highly sensitive to the pharmacological inhibition of splicing. The discovery of these alterations in RNA processing has not only provided new insights into cancer pathogenesis but identified novel therapeutic vulnerabilities and therapeutic opportunities in targeting these aberrations in various ways (e.g., small molecules, splice-switching oligonucleotides (SSOs), and protein therapies) to modulate alternative RNA splicing or other RNA processing and modification mechanisms. Some of these strategies are currently progressing toward clinical development or are already in clinical trials. Additionally, tumor-specific neoantigens produced from these pathogenically spliced events and other abnormal RNA processes provide a potentially extensive source of tumor-specific therapeutic antigens (TAs) for targeted cancer immunotherapy. Moreover, a better understanding of the molecular mechanisms associated with aberrant RNA processes and the biological impact they play might provide insights into cancer initiation, progression, and metastasis. Our goal is to highlight key alternative RNA splicing and processing mechanisms and their roles in cancer pathophysiology as well as emerging therapeutic alternative splicing targets in cancer, particularly in gastrointestinal (GI) malignancies.

The Expanding Role of Alternative Splicing in Vascular Smooth Muscle Cell Plasticity.

Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to 'switch' between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation.

Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.

Chromatin-mediated alternative splicing regulates cocaine-reward behavior.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Transcriptome-based insights into gene networks controlling myopia prevention.

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.

Progesterone receptor antagonists reverse stem cell expansion and the paracrine effectors of progesterone action in the mouse mammary gland.

BACKGROUND: The ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation. A specific consequence of progesterone exposure is the expansion of the mammary stem cell (MSC) and luminal progenitor (LP) compartments. We hypothesized that this effect, and its molecular facilitators, could be abrogated by progesterone receptor (PR) antagonists administered in a mouse model. METHODS: Ovariectomized FVB mice were randomized to 14 days of treatment: sham, EP, EP + telapristone (EP + TPA), EP + mifepristone (EP + MFP). Mice were then sacrificed, mammary glands harvested, and mammary epithelial cell lineages separated by flow cytometry using cell surface markers. RNA from each lineage was sequenced and differential gene expression was analyzed using DESeq. Quantitative PCR was performed to confirm the candidate genes discovered in RNA seq. ANOVA with Tukey post hoc analysis was performed to compare relative expression. Alternative splicing events were examined using the rMATs multivariate analysis tool. RESULTS: Significant increases in the MSC and luminal mature (LM) cell fractions were observed following EP treatment compared to control (p < 0.01 and p < 0.05, respectively), whereas the LP fraction was significantly reduced (p < 0.05). These hormone-induced effects were reversed upon exposure to TPA and MFP (p < 0.01 for both). Gene Ontology analysis of RNA-sequencing data showed EP-induced enrichment of several pathways, with the largest effect on Wnt signaling in MSC, significantly repressed by PR inhibitors. In LP cells, significant induction of Wnt4 and Rankl, and Wnt pathway intermediates Lrp2 and Axin2 (confirmed by qRTPCR) were reversed by TPA and MFP (p < 0.0001). Downstream signaling intermediates of these pathways (Lrp5, Mmp7) showed similar effects. Expression of markers of epithelial-mesenchymal transition (Cdh1, Cdh3) and the induction of EMT regulators (Zeb1, Zeb2, Gli3, Snai1, and Ptch2) were significantly responsive to progesterone. EP treatment was associated with large-scale alternative splicing events, with an enrichment of motifs associated with Srsf, Esrp, and Rbfox families. Exon skipping was observed in Cdh1, Enah, and Brd4. CONCLUSIONS: PR inhibition reverses known tumorigenic pathways in the mammary gland and suppresses a previously unknown effect of progesterone on RNA splicing events. In total, our results strengthen the case for reconsideration of PR inhibitors for breast cancer prevention.

Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. 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 Xon, does not require the co-expression of any regulatory proteins. Using Xon, 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 Xon 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 Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.

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.

Oncogenic dysregulation of pre-mRNA processing by protein kinases: challenges and therapeutic opportunities.

Alternative splicing and polyadenylation represent two major steps in pre-mRNA-processing, which ensure proper gene expression and diversification of human transcriptomes. Deregulation of these processes contributes to oncogenic programmes involved in the onset, progression and evolution of human cancers, which often result in the acquisition of resistance to existing therapies. On the other hand, cancer cells frequently increase their transcriptional rate and develop a transcriptional addiction, which imposes a high stress on the pre-mRNA-processing machinery and establishes a therapeutically exploitable vulnerability. A prominent role in fine-tuning pre-mRNA-processing mechanisms is played by three main families of protein kinases: serine arginine protein kinase (SRPK), CDC-like kinase (CLK) and cyclin-dependent kinase (CDK). These kinases phosphorylate the RNA polymerase, splicing factors and regulatory proteins involved in cleavage and polyadenylation of the nascent transcripts. The activity of SRPKs, CLKs and CDKs can be altered in cancer cells, and their inhibition was shown to exert anticancer effects. In this review, we describe key findings that have been reported on these topics and discuss challenges and opportunities of developing therapeutic approaches targeting splicing factor kinases.

Second-generation antipsychotics induce cardiotoxicity by disrupting spliceosome signaling: Implications from proteomic and transcriptomic analyses.

Second-generation antipsychotics (SGAs) are first-line drugs that are prescribed for mental disorders in clinic. Severe cardiotoxicity has been widely reported and thus limits their clinical application. This study aimed to identify the common mechanism underlying SGAs-induced cardiotoxicity using dual-omics analyses. Balb/C mice were intraperitoneally injected with two representative SGAs, olanzapine (2.5 mg/kg) and clozapine (25 mg/kg), at clinically comparable doses for 0, 7, 14 and 21 days. Our results showed that both SGAs induced cardiomyocyte degeneration, inflammation infiltration, and cardiac fibrosis, all of which worsened with time. Proteomic analysis revelaed that 22 differentially expressed (DE) proteins overlapped in olanzapine and clozapine-treated hearts. These proteins were significantly enriched in muscle contraction, amino acid metabolism and spliceosomal assembly by GO term analysis and spliceosome signaling was among the top enriched pathways by KEGG analysis. Among the 22 DE proteins, three spliceosome signal proteins were validated in a dynamic detection, and their expression significantly correlated with the extent of SGAs-induced cardiac fibrosis. Following the spliceosome signaling dysregulation, RNA sequencing revealed that alternative splicing events in the mouse hearts were markedly enhanced by SGAs treatments, and the production of vast transcript variants resulted in dysregulation of multiple pathways that are critical for cardiomyocytes adaptation and cardiac remodeling. Pladienolide B, a specific inhibitor of mRNA splicing, successfully corrected SGAs-induced alternative splicing and significantly attenuated the secretion of pro-inflammatory factors and cell deaths induced by SGAs exposure. Our study concluded that the spliceosome signaling was a common pathway driving SGAs cardiotoxicity. Pharmacological inhibition of the spliceosome signaling represents a novel therapeutic strategy against SGAs cardiotoxicity.

Detection of alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio Iso-Seq.

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.

Glucocorticoid Receptor ß (GRß): Beyond Its Dominant-Negative Function.

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRß, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRß has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRß actions are restricted to its dominant-negative effects on GRα-mediated responses, GRß has been shown to have intrinsic activities and "directly" regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRß has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRß-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRß and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans.

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.