Changes In Splicing Research Articles

The Estrogen Receptor α Signaling Pathway Controls Alternative Splicing in the Absence of Ligands in Breast Cancer Cells.

Simple SummaryAberrant alternative splicing is now considered a hallmark of cancer, including breast cancer. This results in the production of novel tumor-specific splice RNA variants, and the activation of biological processes such as epithelial-to-mesenchymal transition, leading to more aggressive phenotypes. The purpose of this study was to explore the role of estrogen receptor α in regulating the expression of RNA-binding proteins in luminal breast cancer cells and to determine the effects of its downregulation at the isoform level by exploring changes in isoform usage and alternative splicing. The findings of this study unravel a novel layer of gene regulation mediated by estrogen receptor α, which is fundamental for breast cancer cell growth as well as epithelial-to-mesenchymal transition. Finally, we foresee that this novel feature should be considered when studying the functional roles of estrogen receptor α in the onset and progression of breast cancer.Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. Methods: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. Results: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3′end processing. ApoERα depletion results in 758 isoform switching events with effects on 3′end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. Conclusion: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level.

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.

Transcriptome-Wide Analyses Identify Dominant as the Predominantly Non-Conservative Alternative Splicing Inheritance Patterns in F1 Chickens.

Transcriptome analysis has been used to investigate many economically traits in chickens; however, alternative splicing still lacks a systematic method of study that is able to promote proteome diversity, and fine-tune expression dynamics. Hybridization has been widely utilized in chicken breeding due to the resulting heterosis, but the dynamic changes in alternative splicing during this process are significant yet unclear. In this study, we performed a reciprocal crossing experiment involving the White Leghorn and Cornish Game chicken breeds which exhibit major differences in body size and reproductive traits, and conducted RNA sequencing of the brain, muscle, and liver tissues to identify the inheritance patterns. A total of 40 515 and 42 612 events were respectively detected in the brain and muscle tissues, with 39 843 observed in the liver; 2807, 4242, and 4538 events significantly different between two breeds were identified in the brain, muscle, and liver tissues, respectively. The hierarchical cluster of tissues from different tissues from all crosses, based on the alternative splicing profiles, suggests high tissue and strain specificity. Furthermore, a comparison between parental strains and hybrid crosses indicated that over one third of alternative splicing genes showed conserved patterns in all three tissues, while the second prevalent pattern was non-additive, which included both dominant and transgressive patterns; this meant that the dominant pattern plays a more important role than suppression. Our study provides an overview of the inheritance patterns of alternative splicing in layer and broiler chickens, to better understand post-transcriptional regulation during hybridization.

Genome-Wide Analysis of Alternative Splicing during Host-Virus Interactions in Chicken.

The chicken is a model animal for the study of evolution, immunity and development. In addition to their use as a model organism, chickens also represent an important agricultural product. Pathogen invasion has already been shown to modulate the expression of hundreds of genes, but the role of alternative splicing in avian virus infection remains unclear. We used RNA-seq data to analyze virus-induced changes in the alternative splicing of Gallus gallus, and found that a large number of alternative splicing events were induced by virus infection both in vivo and in vitro. Virus-responsive alternative splicing events preferentially occurred in genes involved in metabolism and transport. Many of the alternatively spliced transcripts were also expressed from genes with a function relating to splicing or immune response, suggesting a potential impact of virus infection on pre-mRNA splicing and immune gene regulation. Moreover, exon skipping was the most frequent AS event in chickens during virus infection. This is the first report describing a genome-wide analysis of alternative splicing in chicken and contributes to the genomic resources available for studying host–virus interaction in this species. Our analysis fills an important knowledge gap in understanding the extent of genome-wide alternative splicing dynamics occurring during avian virus infection and provides the impetus for the further exploration of AS in chicken defense signaling and homeostasis.

Developing biomarkers of TDP‐43‐related splicing dysfunction in frontotemporal dementia

AbstractBackgroundFrontotemporal dementia (FTD) is a rapidly progressive neurodegenerative disorder characterized by prominent atrophy of the frontal and temporal lobes and marked language and/or behavioral disturbances. Mislocalization of the TAR DNA‐binding protein 43 (TDP‐43) – characterized by both its nuclear loss and its cytosolic accumulation – occurs in up to 50% of FTD cases. Recent evidence from our group and others demonstrates that loss of TDP‐43‐dependent splicing activity occurs in FTD patient brains, is an early disease phenomenon, and contributes directly to neuronal death. Monitoring the products of TDP‐43 related mis‐splicing may, thus, yield promising biomarker candidates of early cellular dysfunction in FTD.MethodWe propose a series of proteogenomic studies in human iPSC neurons, post‐mortem brain, and patient biospecimens to identify and develop protein‐based biomarkers of RNA mis‐splicing in FTD. First, we will identify pathologic RNA splicing changes and protein products of mis‐splicing that occur in the context of TDP‐43 depletion in iPSC‐derived neurons. We will next determine which of these predicted transcripts and de novo proteins occur in post‐mortem human FTD brains, as a crucial first step in biomarker development. Finally, we will develop CSF biomarkers of FTD‐related mis‐splicing through a combination of targeted proteomics and ELISA‐based assays of lead candidate biomarkers.ResultUsing CRISPRi knockdown of TDP‐43 in i3Neurons followed by total RNAseq, we detected cryptic exon formation in STMN2 as well as hundreds of additional transcripts. We developed a proteogenomic informatic pipeline to detect de novo protein products of mis‐spliced transcripts in TDP‐43 deficient i3Neurons, revealing two in‐frame de novo peptides in the MYO18A transcript. Furthermore, we detected 38 of these mis‐spliced transcripts in ALS/FTD post‐mortem cortex, including MYO18A.ConclusionThese preliminary studies indicate that our i3Neuron CRISPRi pipeline can predict disease‐relevant markers of TDP‐43 mis‐splicing and suggest a proteogenomic workflow for development of novel protein‐based biomarkers of mis‐spliced transcripts.Together, these efforts will yield early biomarkers of TDP‐43 related cellular dysfunction that can act as surrogate measures of disease onset and progression in FTD.

Abstract P170: SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.

Abstract 13426: CRISPR Guide RNA Design is Able to Use for Studying Splicing and Circadian Gene Expression

Introduction: CRISPR editing of genomic regulatory elements with the intention of direct exon/intron splicing is a hopeful means of everlastingly alleviating genetic diseases. Cas9-induced DNA double-stranded break, non-homologous end-joining repair pathways frequently introduce variable indel mutations at the cut site. Circadian rhythms control cell proliferation and differentiation, however circadian control of Human induced pluripotent stem cells (hiPSCs) remains study at the molecular level. Hypothesis: Elucidation of circadian rhythms in hiPSCs differentiated cardiomyocytes. Methods: In the current investigation, hiPSCs carrying knockout mutations for Per1, Per2, and connexin43 were generated via CRISPR/Cas9 gene-editing technology. hiPSCs were differentiated into cardiomyocytes (CM) and spheroid cultured was performed as previously described. We evaluated different guide RNAs Cas9-induced DNA double-stranded break were analyzed using Sanger sequencing and bioinformatics. Optical mapping was used to measure the electrical properties of individual spheroids and monolayers of WT, connexin 43 KO, and overexpression of connexin 43 (WT, Cx43 KO, and Cx43 OV). We also study in the hiPSCs CMs of monolayer resistivity and reactivity and compared them with rabbit ventricular myocardium. Results: To determine the genomic regulatory elements that control exon splicing we designed several guides Per1, Per2, and Connexin 43. Our sequencing and bioinformatics analysis found untranslated region or coding region designed guide RNA found changes in splicing but protein truncation observed in connexin 43 of the coding region. To further understand the differential expression of Cx43 in hiPSC-CMs optical mapping in monolayers and spheroids were observed decreased conduction velocities. To further understand we also perform impedance measurement in WT, Cx43 KO, and Cx43-OV. We found resistivity and reactivity were significantly changed between groups. We will look these mechanisms has any role in the spheroid treatment is associated with significant changes in arrhythmogenicity of swine studies. Conclusions: Our study established the CRISPR gRNA design can be used study splicing and circadian pattern regulated by connexin 43.

The Efficacy of Whole Genome Sequencing and RNA-Seq in the Diagnosis of Whole Exome Sequencing Negative Patients with Complex Neurological Phenotypes.

Whole-genome sequencing (WGS) is being increasingly utilized for the diagnosis of neurological disease by sequencing both the exome and the remaining 98 to 99% of the genetic code. In addition to more complete coverage, WGS can detect structural variants (SVs) and intronic variants (SNVs) that cannot be identified by whole exome sequencing (WES) or chromosome microarray (CMA). Other multi-omics tools, such as RNA sequencing (RNA-Seq), can be used in conjunction with WGS to functionally validate certain variants by detecting changes in gene expression and splicing. The objective of this retrospective study was to measure the diagnostic yield of duo/trio-based WGS and RNA-Seq in a cohort of 22 patients (20 families) with pediatric onset neurological phenotypes and negative or inconclusive WES results in lieu of reanalysis. WGS with RNA-Seq resulted in a definite diagnosis of an additional 25% of cases. Sixty percent of these solved cases arose from the identification of variants that were missed by WES. Variants that could not be unequivocally proven to be causative of the patients' condition were identified in an additional 5% of cases.

What sequencing technologies can teach us about innate immunity.

For years, we have taken a reductionist approach to understanding gene regulation through the study of one gene in one cell at a time. While this approach has been fruitful it is laborious and fails to provide a global picture of what is occurring in complex situations involving tightly coordinated immune responses. The emergence of whole-genome techniques provides a system-level view of a response and can provide a plethora of information on events occurring in a cell from gene expression changes to splicing changes and chemical modifications. As with any technology, this often results in more questions than answers, but this wealth of knowledge is providing us with an unprecedented view of what occurs inside our cells during an immune response. In this review, we will discuss the current RNA-sequencing technologies and what they are helping us learn about the innate immune system.

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting synthetic lethality of homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Thus, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.We have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and MV-4-11), CTX-712 showed a strong inhibitory effect on cell proliferation (IC 50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 primary AML cells (the average of IC 50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF3/4/6, all of which bind to SRSF2. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in skipped exon. Notably, the degree of splicing (percent spliced-in value) in skipped exon events induced by the drug was positively correlated with the sensitivity to the drug (IC 50) in primary AML cells (n=32, R=0.61, P=0.00018).To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 MDS/AML-derived xenografts (PDXs), which were treated with varying doses of CTX-712. Among the 13 PDX models, SRSF2 mutation was found in 2 cases, which had the SRSF2 P95H or P95L mutation. The SRSF2 P95H PDX showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm 3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.0395), and 39 ± 39 (high dose, P=0.0064) (N=5 each, mean ± SEM). Interestingly CLK inhibition induced aberrant splicing events including skipped exons in vivo, which were more strongly affected in the SRSF2-mutated model. In addition, CTX-712 efficacy was also confirmed in the model with the SRSF2 P95L mutation. The SRSF2 P95L model showed a significant reduction in tumor volumes (mm 3) 2 weeks after CTX-712 treatment; 406 ± 94 (vehicle) and 75 ± 17 (high dose, P=0.0162) (N=6 each, mean ± SEM). CTX-712 also significantly improved the survival of the SRSF2 P95L-mutated model (high dose, P=0.0030) (N=6 each).Overall, 10 out of 13 PDX AML/MDS models, including 2 SRSF2-mutated models, showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in the SRSF2 P95H mutation model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. DisclosuresYoda: Chordia Therapeutics Inc.: Research Funding. Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Mizutani: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Tozaki: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Satoh: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Miyake: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila.

SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

SummaryAlternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.

Experimental Design for Time-Series RNA-Seq Analysis of Gene Expression and Alternative Splicing.

RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.

MDM4 Isoform Expression in Melanoma Supports an Oncogenic Role for MDM4-A.

The p53 tumor suppressor integrates upstream signals such as DNA damage and active oncogenes to initiate cell cycle arrest or apoptosis. This response is critical to halting inappropriate growth signals. As such, p53 activity is lost in cancer. In melanoma, however, the p53 gene is intact in a reported 94% of human cases. Rather than direct mutation, p53 is held inactive through interaction with inhibitory proteins. Here, we examine the expression of the two primary inhibitors of p53, MDM2 and MDM4, in genomic databases and biopsy specimens. We find that MDM4 is frequently overexpressed. Moreover, changes in splicing of MDM4 occur frequently and early in melanomagenesis. These changes in splicing must be considered in the design of therapeutic inhibitors of the MDM2/4 proteins for melanoma.

Biallelic TMEM260 variants cause truncus arteriosus, with or without renal defects.

Only two families have been reported with biallelic TMEM260 variants segregating with structural heart defects and renal anomalies syndrome (SHDRA). With a combination of genome, exome sequencing and RNA studies, we identified eight individuals from five families with biallelic TMEM260 variants. Variants included one multi-exon deletion, four nonsense/frameshifts, two splicing changes and one missense change. Together with the published cases, analysis of clinical data revealed ventricular septal defects (12/12), mostly secondary to truncus arteriosus (10/12), elevated creatinine levels (6/12), horse-shoe kidneys (1/12) and renal cysts (1/12) in patients. Three pregnancies were terminated on detection of severe congenital anomalies. Six patients died between the ages of 6 weeks and 5 years. Using a range of stringencies, carrier frequency for SHDRA was estimated at 0.0007-0.007 across ancestries. In conclusion, this study confirms the genetic basis of SHDRA, expands its known mutational spectrum and clarifies its clinical features. We demonstrate that SHDRA is a severe condition associated with substantial mortality in early childhood and characterised by congenital cardiac malformations with a variable renal phenotype.

P-089: Identification of novel targets in multiple myeloma for “undruggable” RAS/CDK signaling cascade

<h3>Background</h3> RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after RAS/CDK inhibition to identify novel molecular targets. <h3>Method</h3> in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC<0.5) MM cytotoxicity triggered by Ei+Ci treatment, which in a dose-dependent manner arrested MM cells in G0/G1 phase and activated mitochondrial apoptotic signaling. Ei+Ci treatment decreased Erk1/2, CDK4/6, and p-Erk1/2 levels in MM cells, and also induced inhibition of key targets (c-myc, p-RSK,-RB, E2F1) of RAS/CDK cascade. Our studies in patient samples indicate that MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that Ei+Ci combination can overcome the protective effects of the MM BM milieu. An in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. Our studies therefore suggesting on-target activity of these inhibitors in vitro/vivo. <h3>Results</h3> We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown and downregulation due to treatment with Ei+Ci were considered spliced gene signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p<2e-11) upregulation of both genes in clonal PCs with progression from MGUS to sMM, and to overt MM. SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 has a trend toward poor outcomes. SNRPB proteins are also overexpressed in MM cells. Evaluating SNRPB effects on RNA splicing showed upregulation of transcripts with full intron retention or transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. Thus, SNRPB overexpression contributes to aberrant transcriptome splicing associated with RAS/CDK cascade in MM. <h3>Conclusions</h3> Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.

The pattern of DNA methylation alteration, and its association with the changes of gene expression and alternative splicing during phosphate starvation in tomato

SUMMARYDNA methylation is changed and associates with gene expression alterations in plant response to phosphate starvation (Pi–), a common stress that impacts plant growth and productivity. However, in the horticultural model species Solanum lycopersicum (tomato), the dynamics of DNA methylation and its relationship with changes in gene transcription and alternative splicing (AS) under Pi– are unknown. Here, we performed integrative methylome and transcriptome analyses of tomato seedlings under Pi‐deficient and ‐sufficient conditions. We found Pi– caused a slight increase in the overall methylation level, with millions of differentially methylated cytosines (DmCs) and a few hundred differentially methylated regions (DMRs). We also identified thousands of differentially expressed (DE) and differential AS (DAS) genes induced by Pi–, and found that DmCs were more abundant in non‐expressed genes than in DE or DAS genes. Moreover, DNA methylation alterations weakly correlated with transcription changes but not with DAS events, and hyper‐CHH‐DMRs overlapping with transposable elements (TEs) were enriched in a subset of Pi starvation response (PSR) genes. We propose that changes in DNA methylation may be associated with the differential expression of some PSR genes, but that most of these changes probably control the expression of nearby TEs, rather than directly affecting the transcription or AS of PSR genes. Besides, the pattern of methylation changes upon Pi– may largely be shaped by TE distributions. Together, our study provides comprehensive insights into the association of DNA methylation with gene transcription and AS under Pi– in tomato and may contribute to unveiling novel roles of epigenetic mechanisms in plant stress response.

Nuclear speckle specific hnRNP D-like prevents age- and AD-related cognitive decline by modulating RNA splicing

BackgroundAberrant alternative splicing plays critical role in aging and age-related diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) reportedly regulate RNA splicing process. Whether and how hnRNPs contribute to age-related neurodegenerative diseases, especially Alzheimer’s disease (AD), remain elusive.MethodsImmunoblotting and immunostaining were performed to determine expression patterns and cellular/subcellular localization of the long isoform of hnRNP D-like (L-DL), which is a hnRNP family member, in mouse hippocampus. Downregulation of L-DL in WT mice was achieved by AAV-mediated shRNA delivery, followed by memory-related behavioural tests. L-DL interactome was analysed by affinity-precipitation and mass spectrometry. Alternative RNA splicing was measured by RNA-seq and analyzed by bioinformatics-based approaches. Downregulation and upregulation of L-DL in APP/PS1 mice were performed using AAV-mediated transduction.ResultsWe show that L-DL is specifically localized to nuclear speckles. L-DL levels are decreased in the hippocampus of aged mouse brains and downregulation of L-DL impairs cognition in mice. L-DL serves as a structural component to recruit other speckle proteins, and regulates cytoskeleton- and synapse-related gene expression by altering RNA splicing. Mechanistically, these splicing changes are modulated via L-DL-mediated interaction of SF3B3, a core component of U2 snRNP, and U2AF65, a U2 spliceosome protein that guides U2 snRNP’s binding to RNA. In addition, L-DL levels are decreased in APP/PS1 mouse brains. While downregulation of L-DL deteriorates memory deficits and overexpression of L-DL improves cognitive function in AD mice, by regulating the alternative splicing and expression of synaptic gene CAMKV.ConclusionsOur findings define a molecular mechanism by which hnRNP L-DL regulates alternative RNA splicing, and establish a direct role for L-DL in AD-related synaptic dysfunction and memory decline.

The HTLV-1 viral oncoproteins Tax and HBZ reprogram the cellular mRNA splicing landscape.

Viral infections are known to hijack the transcription and translation of the host cell. However, the extent to which viral proteins coordinate these perturbations remains unclear. Here we used a model system, the human T-cell leukemia virus type 1 (HTLV-1), and systematically analyzed the transcriptome and interactome of key effectors oncoviral proteins Tax and HBZ. We showed that Tax and HBZ target distinct but also common transcription factors. Unexpectedly, we also uncovered a large set of interactions with RNA-binding proteins, including the U2 auxiliary factor large subunit (U2AF2), a key cellular regulator of pre-mRNA splicing. We discovered that Tax and HBZ perturb the splicing landscape by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia/lymphoma (ATLL) samples from two independent patient cohorts, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinated by Tax and HBZ to reprogram the transcriptome.

Do Fragile X Syndrome and Other Intellectual Disorders Converge at Aberrant Pre-mRNA Splicing?

Fragile X Syndrome is a neuro-developmental disorder caused by the silencing of the FMR1 gene, resulting in the loss of its protein product, FMRP. FMRP binds mRNA and represses general translation in the brain. Transcriptome analysis of the Fmr1-deficient mouse hippocampus reveals widespread dysregulation of alternative splicing of pre-mRNAs. Many of these aberrant splicing changes coincide with those found in post-mortem brain tissue from individuals with autism spectrum disorders (ASDs) as well as in mouse models of intellectual disability such as PTEN hamartoma syndrome (PHTS) and Rett Syndrome (RTT). These splicing changes could result from chromatin modifications (e.g., in FXS, RTT) and/or splicing factor alterations (e.g., PTEN, autism). Based on the identities of the RNAs that are mis-spliced in these disorders, it may be that they are at least partly responsible for some shared pathophysiological conditions. The convergence of splicing aberrations among these autism spectrum disorders might be crucial to understanding their underlying cognitive impairments.