Splicing Reporter Research Articles

The C. elegans truncated insulin receptor DAF-2B regulates survival of L1 arrested larvae.

We have previously characterized a truncated isoform of the C. elegans insulin-like receptor, DAF-2B, which retains the ligand binding domain but cannot transduce a signal due to the absence of the intracellular signaling domain. DAF-2B modifies insulin / insulin-like growth factor signaling-dependent processes, such as dauer formation and lifespan, by sequestering insulin-like peptides (ILP) and preventing signaling through full length DAF-2 receptors. Here we show that DAF-2B is also important for starvation resistance, as genetic loss of daf-2b reduces survival in arrested first stage larvae (L1). Under fed conditions, we observe daf-2b splicing capacity in both the intestine and the hypodermis, but in starved L1s this becomes predominantly hypodermal. Using a novel splicing reporter system, we observe an increase in the ratio of truncated to full length insulin receptor splicing capacity in starved L1 larvae compared with fed, that may indicate a decrease in whole body insulin responsiveness. Consistent with this, overexpression of DAF-2B from the hypodermis, but not the intestine, confers increased survival to L1 animals under starvation conditions. Our findings demonstrate that the truncated insulin receptor DAF-2B is involved in the response to L1 starvation and promotes survival when expressed from the hypodermis.

Decreased Embryonic Splicing Repressor Expression Enhances Splicing Modulator Sensitivity in Pediatric Acute Myeloid Leukemia Stem Cells

Adults with myeloid malignancies frequently harbor mutations in splicing regulatory genes, such as SRSF2, U2AF1, and SF3B1. In pediatric AML (pAML), splicing factor mutations are relatively infrequent in bulk populations of cells. However, by performing whole exome sequencing, single cell proteogenomics (Tapestri®) as well as whole transcriptome RNA sequencing (RNA-seq) analyses of FACS-purified hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we discovered an increased frequency of exon skipping, typical of embryonic splicing patterns, in pAML HSCs and HPCs. By employing single cell proteogenomics (Tapestri®) analysis, we discovered SF3B1 splicing factor mutations at the single cell in pAML CD34+ cells that were not readily detectable by whole exome sequencing in bulk populations thereby suggesting that clonal expansion of minor clones may fuel leukemic stem cell (LSC) generation. Further RNA-seq analysis demonstrated that both pAML HSCs and HPCs harbored decreased expression of a repressor of embryonic splicing, RNA-binding Fox protein 2, RBFOX2(also known as RBM9). By employing, rMATS, an algorithm that detects splicing events, we discovered differential splicing of genes involved in cell cycle regulation, DNA repair and chromatin organization in pAML more than in non-leukemic (NL) samples. In addition, differentially spliced RBFOX2 target genes were found to be involved in mRNA processing. In pAML HSCs, we observed differential splicing of THOC7, which regulates mRNA 3' end processing while in pAML HPCs, we uncovered a cluster of splicing regulators, such as SRSF7, SRSF11 and U2AF2. Thus, pAML is typified by widespread splicing deregulation in the setting of RBFOX2 downregulation. Previous reports suggest that splicing deregulation represents a therapeutic vulnerability in a broad array of malignancies. Thus, we examined the response of pAML cell lines in vitro and in lentiviral splicing reporter humanized AML mouse models to Rebecsinib (17S-FD-895; Crews et al, Cell Stem Cell, 2016), a small molecule splicing modulator that is currently undergoing IND enabling studies. In these studies, Rebecsinib treatment reduced pAML cell survival at doses that altered splicing reporter activity and spared normal hematopoietic stem and progenitor cells (HSPCs). Notably, shRNA knockdown of RBFOX2 in cord blood CD34+ HSPCs phenocopied aspects of pAML in that it significantly increased clonogenic capacity and sensitivity to Rebecsinib in replating assays. In summary, decreased RBFOX2 expression and reversion to an embryonic splicing program typified by differential exon usage in pAML HSPCs was associated with increased sensitivity to selective splicing modulation and may represent a new therapeutic paradigm for pediatric patients with relapsed or refractory AML.

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.

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions.

Spliced leader trans splicing is the addition of a short, capped sequence to the 5' end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5' trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target. We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor.

I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model.Key messages• Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified.• A splicing reporter assay demonstrated that the variant affected the TTN splicing.• Rbm20I538T mice showed neither DCM nor cardiac dysfunction.• Rbm20I538T mice showed different splicing patterns and the gene expressions.

KIRA8 attenuates non-alcoholic steatohepatitis through inhibition of the IRE1α/XBP1 signalling pathway

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

Brief Summary: This study reports splice-disruptive variants in POU1F1 in 4 families with hypopituitarism and uses a high-throughput splicing reporter assay to create a comprehensive catalogue of such variants in or near exon 2 of the gene. The catalogue paves the way for identifying synonymous but splice-disruptive POU1F1 variants in additional patients with hypopituitarism, which underscores the importance of evaluating defective splicing as a disease mechanism.

Modeling splicing outcome by combining 5'ss strength and splicing regulatory elements.

Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5′ss and 3′ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parallel splicing reporter assay on more than 3000 sequences and exemplarily identified some SRE binding proteins. Aiming at a unified ‘functional splice site strength’ encompassing both U1 snRNA complementarity and impact from neighboring SREs, we developed a novel RNA-seq based 5′ss usage landscape, mapping the competition of pairs of high confidence 5′ss and neighboring exonic GT sites along HBond and HEXplorer score coordinate axes on human fibroblast and endothelium transcriptome datasets. These RNA-seq data served as basis for a logistic 5′ss usage prediction model, which greatly improved discrimination between strong but unused exonic GT sites and annotated highly used 5′ss. Our 5′ss usage landscape offers a unified view on 5′ss and SRE neighborhood impact on splice site recognition, and may contribute to improved mutation assessment in human genetics.

ACT1-CUP1 Assays Determine the Substrate-specific Sensitivities of Spliceosomal Mutants in Budding Yeast.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on Survival Motor Neuron 2 (SMN2) mRNA Splicing and Expression.

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the survival motor neuron 1 (SMN1) gene. SMN1 is duplicated in humans to give rise to the paralogous survival motor neuron 2 (SMN2) gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of SMN2 transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since SMN2 copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate SMN2 expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in SMN2 splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including TIA1 and FABP3, for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases SMN2 expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of SMN2 in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length SMN2 mRNAs by targeting TIA1 and FABP3 expression, which is distinct from other small molecule regulators of SMN2 alternative splicing. This study provides a novel means to increase full-length SMN2 expression and a novel target for therapeutics development.

Translational suppression via IFG-1/eIF4G inhibits stress-induced RNA alternative splicing in Caenorhabditis elegans.

Splicing of precursor mRNA is an essential process for dividing cells, and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to modify alternative splicing, and molecular mechanisms that influence stress-induced alternative splicing remain unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved eukaryotic initiation factor 4G (IFG-1/eIF4G) inhibits cadmium-induced alternative splicing. Transcriptome analysis of an ifg-1-deficient mutant revealed an overall decrease in intronic and intergenic reads and prevented cadmium-induced alternative splicing compared to the wild type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Depletion of snr-1 and snr-2 also diminished the enhanced cadmium resistance of the ifg-1 mutant. Together, these data describe a molecular mechanism through which translation suppression inhibits stress-induced alternative splicing and demonstrate an essential role for RNA splicing in promoting longevity and stress resistance in a translation-compromised mutant.

Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity.

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5’ splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.

A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts

Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.

Tissue-specific expression of myosin phosphatase subunits and isoforms in smooth muscle of mice and humans.

Alternative splicing of exon24 (E24) of myosin phosphatase targeting subunit 1 (Mypt1) by setting sensitivity to nitric oxide (NO)/cGMP-mediated relaxation is a key determinant of smooth muscle function. Here we defined expression of myosin phosphatase (MP) subunits and isoforms by creation of new genetic mouse models, assay of human and mouse tissues, and query of public databases. A Mypt1-LacZ reporter mouse revealed that Mypt1 transcription is turned on early in development during smooth muscle differentiation. Mypt1 is not as tightly restricted in its expression as smooth muscle myosin heavy chain (Myh11) and its E6 splice variant. Mypt1 is enriched in mature smooth versus nonmuscle cells. The E24 splice variant and leucine zipper minus protein isoform that it encodes is enriched in phasic versus tonic smooth muscle. In the vascular system, E24 splicing increases as vessel size decreases. In the gastrointestinal system, E24 splicing is most predominant in smooth muscle of the small intestine. Tissue-specific expression of MP subunits and Mypt1 E24 splicing is conserved in humans, whereas a splice variant of the inhibitory subunit (CPI-17) is unique to humans. A Mypt1 E24 mini-gene splicing reporter mouse generated to define patterns of E24 splicing in smooth muscle cells (SMCs) dispersed throughout the organ systems was unsuccessful. In summary, expression of Mypt1 and splicing of E24 is part of the program of smooth muscle differentiation, is further enhanced in phasic smooth muscle, and is conserved in humans. Its low-level expression in nonmuscle cells may confound its measurement in tissue samples.

Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

PALB2 loss‐of‐function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice‐site variants detected in approximately 113,000 women in the large‐scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty‐two PALB2 variants at the intron‐exon boundaries were analyzed with MaxEntScan. Forty‐two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1–12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site‐directed mutagenesis and assayed in MCF‐7/MDA‐MB‐231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full‐length transcripts of each minigene. More than 30 different variant‐induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in‐house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG‐AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49‐2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in‐frame transcripts of unknown impact on the PALB2 protein function or the minigene full‐length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG‐AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient.

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

Abstract 10966: Characterization of the Novel Cardiac Splicing Factor Slm2 in Heart Failure, Which Regulates Splicing of Titin

Introduction: The versatility of the human genome is increased by the process of alternative mRNA splicing. Impaired splicing of the cardiac transcriptome is involved in the pathophysiology of heart failure. Especially, mutations in cardiac-specific splicing factors such as RBM20 cause severe forms of cardiomyopathy. Aim: We aimed to identify novel cardiomyopathy-associated splicing factors in the human heart using a score of myocardial tissue specificity including 53 human tissues and disease-associated expression changes in hearts of dilated cardiomyopathy (DCM) patients and controls. Methods & Results: We found the splicing factor Slm2 to be significantly upregulated on the mRNA and protein level in the human myocardium of patients diagnosed with DCM (1.88 and 5.3-fold, both P<0.05). Reduction of Slm2 in vivo resulted in DCM in zebrafish and sarcomere irregularity and altered calcium cycling in human cardiomyocytes. Sequencing of RNAs bound to Slm2 isolated from diseased human hearts, revealed the interaction of Slm2 with important mRNA transcripts encoding for sarcomere constituents, such as troponin I, troponin T, tropomyosin and titin. In the failing human myocardium, Slm2 bound to an alternative titin mRNA variant including retained introns. In detail, Slm2 interacted with exons of the titin mRNA encoding for the PEVK region, which mediates flexibility and contractile properties of the heart muscle. Using a splice reporter assay, which contains parts of titin’s PEVK region, we found Slm2 mediating the splicing process of intron retention. Conclusion: Slm2 is a novel splicing factor in the diseased human myocardium, maintaining cardiomyocyte integrity and function by binding and splicing essential sarcomere constituents including titin.

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.

Deregulation of Splicing in Pediatric Acute Myeloid Stem and Progenitor Cells

-Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study.Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation.After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal.Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence. DisclosuresCloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Biallelic FRA10AC1 variants cause a neurodevelopmental disorder with growth retardation.

The major spliceosome mediates pre-mRNA splicing by recognizing the highly conserved sequences at the 5' and 3' splice sites and the branch point. More than 150 proteins participate in the splicing process and are organized in the spliceosomal A, B, and C complexes. FRA10AC1 is a peripheral protein of the spliceosomal C complex and its ortholog in the green alga facilitates recognition or interaction with splice sites. We identified biallelic pathogenic variants in FRA10AC1 in five individuals from three consanguineous families. The two unrelated Patients 1 and 2 with loss-of-function variants showed developmental delay, intellectual disability, and no speech, while three siblings with the c.494_496delAAG (p.Glu165del) variant had borderline to mild intellectual disability. All patients had microcephaly, hypoplasia or agenesis of the corpus callosum, growth retardation, and craniofacial dysmorphism. FRA10AC1 transcripts and proteins were drastically reduced or absent in fibroblasts of Patients 1 and 2. In a heterologous expression system, the p.Glu165del variant impacts intrinsic stability of FRA10AC1 but does not affect its nuclear localization. By co-immunoprecipitation, we found ectopically expressed HA-FRA10AC1 in complex with endogenous DGCR14, another component of the spliceosomal C complex, while the splice factors CHERP, NKAP, RED, and SF3B2 could not be co-immunoprecipitated. Using an in vitro splicing reporter assay, we did not obtain evidence for FRA10AC1 deficiency to suppress missplicing events caused by mutations in the highly conserved dinucleotides of 5' and 3' splice sites in an in vitro splicing assay in patient-derived fibroblasts. Our data highlight the importance of specific peripheral spliceosomal C complex proteins for neurodevelopment. It remains possible that FRA10AC1 may have other and/or additional cellular functions, such as coupling of transcription and splicing reactions.