Exon Usage Research Articles

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing (NGS) approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden-death medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA expression (9 samples per group). DeSeq2 identified thirty-five genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted p<0.1). In depression, altered genes include humanin like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted p<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted p<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Increased expression levels of ppGalNAc-T13 in lung cancers: Significance in the prognostic diagnosis.

ppGalNAc-T13 is upregulated along with reduced expression of GM1 in high metastatic sublines of the murine Lewis lung cancer cell line, but little is known about the implication of ppGalNAc-T13 expression in human cancers. Since lung cancer cell lines showed high expression levels of ppGalNAc-T13, we analyzed ppGalNAc-T13 expression in surgical lung cancer specimens to examine whether ppGalNAc-T13 can be used as a prognostic marker or a therapeutic target. We analyzed mRNA expression levels of GALNT13 and its variant exon usages in surgical specimens by real-time RT-PCR, and the results were evaluated by correlating with clinical data. Ninety-one surgical specimens were analyzed. Consequently, recurrence-free survival was significantly shorter (P=0.045) in high expression group of GALNT13 mRNA. In the analysis of tumor specific exon usage in GALNT13 RNA sequence, one variant exon was significantly associated with worse prognosis. By contrast, in another variant exon, positive variant expression group showed better prognosis than negative group. We also tried to detect GALNT13 mRNA in 63 serum samples from patients with lung cancers to examine whether GALNT13 mRNA can be measured in body fluids, detecting significant levels in 4 samples. Finally, expression of GM1, ppGalNAc-T13 and trimeric Tn antigen was examined by immunohistochemistry in order to evaluate them as a prognostic factor. It was demonstrated that ppGalNAc-T13 and trimeric Tn antigen had a relationship with worse prognosis in 35 investigated lung cancer patients. In conclusion, our results suggest that ppGalNAc-T13 might be a useful prognostic factor of lung cancers.

Increased THEMIS First Exon Usage in CD4+ T-Cells Is Associated with a Genotype that Is Protective against Multiple Sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system. Genome wide association studies have identified over 100 common variants associated with multiple sclerosis, the majority of which implicate immunologically relevant genes, particularly those involved in T-cell development. SNP rs13204742 at the THEMIS/PTPRK locus is one such variant. Here, we have demonstrated mutually exclusive use of exon 1 and 2 amongst 16 novel THEMIS isoforms. We also show inverse correlation between THEMIS expression in human CD4+ T-cells and dosage of the multiple sclerosis risk allele at rs13204742, driven by reduced expression of exon 1- containing isoforms. In silico analysis suggests that this may be due to cell-specific, allele-dependent binding of the transcription factors FoxP3 and/or E47. Research exploring the functional implications of GWAS variants is important for gaining an understanding of disease pathogenesis, with the ultimate aim of identifying new therapeutic targets.

Abstract LB-289: Hypermethylation enforces promoter switch in glioblastoma multiforme tumors

Abstract Complete understanding why a patient is sensitive or resistant to a treatment needs comprehensive whole-genome characterization of the patient's tumor to address underlying molecular mechanism. The developed consortiums, such as The Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), provide such genomic data at different dimensions (e.g., DNA methylation, RNA gene expression, DNA copy number, and DNA mutation). However, there lacks a patient-specific approach to integration of the multi-dimensional genomic data, which is helpful to provide mechanistic insights for primary or recurrent tumor of an individual patient. Here, we developed a new patient-specific analysis for the genomic data integration and its application to Glioblastoma Multiforme (GBM) tumors discovered a mechanism of epigenetic regulation on drug response of Temozolomide (TMZ). We applied the new patient-specific analysis to 13 recurrent tumors of GBM patients in the TCGA and identified a top gene (#1), CYP19A1, with alternative promoters and intragenic hypermethylation. We detected the gene isoforms of CYP19A1 in the different brain tissues. The whole gene (Exons 1-10) is seldomly expressed in both of the normal brain tissues and low-grade gliomas tumors whereas new gene isoforms are specifically expressed in the primary GBM tumors and recurrent GBM tumors. Among 175 primary GBM tumors, 166 (95%) expressed the isoforms of CYP19A1. In the primary and recurrent GBM tumors, there are 78% (129/166) and 83% (10/12) expressing the isoforms starting from the TSS near to Exon 4 (Ex-4). CYP19A1 has (i) hypermethylated CpGs at the regions of original promoter and the introns upstream of Ex-4, and (ii) alternative exon usage: Exons 4-10 are highly used whereas exons 1-3 are seldomly used. We hypothesize that the hypermethylated CpGs located in the region covering from the promoter (near to TSS) to the intron close to Ex-4 may prevent the start of the TF complex binding to the original promoter and enforce the switch to alternative promoter proximal to Ex-4. The Pol-II ChIP-Seq data of U87 cell line from the ENCODE indicate that the Pol-II alternatively binds to the upstream of Ex-4 because the CpGs close to the original promoters, I.f and I.3, are hypermethylated. We further treated the U87 cell line with 5-AZA to induce demethylation of CpGs. Strikingly, we observed that Ex-2 is highly expressed after demethylation. The results confirmed that the region near to Ex-4 is an alternative promoter for expression of the isoforms of CYP19A1 and the hypermethylation of CpGs controls the usage of Exons 1-10. We tested if the gene expression and new transcript isoforms of CYP19A1 are related to patients’ survival. We checked the gene expression of CYP19A1 in 161 primary tumors of GBM from TCGA, who received radiotherapy, surgery, and chemotherapy of TMZ. The primary tumors of GBM patients with lower CYP19A1 tend to benefit more and survive longer. The statistical significance for the survival analysis based on Cox regression is P &amp;lt; 0.007. We analyzed that the survival statuses of these patients with different gene isoforms. The new isoform starting from Exon 4 to Exon 8 benefits patients’ survival more (628±453), compared to those terminated proximal to Exon 9 (317±152) and those proximal to Exon 10 (367±251). The statistical significance is P = 0.008, Wilcoxon rank sum test. Citation Format: Guangxu Jin, Liang Liu, Xiaobo Zhou. Hypermethylation enforces promoter switch in glioblastoma multiforme tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-289.

MiRNA-Mediated KHSRP Silencing Rewires Distinct Post-transcriptional Programs during TGF-β-Induced Epithelial-to-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) confers several traits to cancer cells that are required for malignant progression. Here, we report that miR-27b-3p-mediated silencing of the single-strand RNA binding protein KHSRP is required for transforming growth factor β (TGF-β)-induced EMT in mammary gland cells. Sustained KHSRP expression limits TGF-β-dependent induction of EMT factors and cell migration, whereas its knockdown inuntreated cellsmimics TGF-β-induced EMT. Genome-wide sequencing analyses revealed that KHSRP controls (1) levels of mature miR-192-5p, a microRNA that targets a group of EMT factors, and (2) alternative splicing of a cohort of pre-mRNAs related to cell adhesion and motility including Cd44 and Fgfr2. KHSRP belongs to a ribonucleoprotein complex that includes hnRNPA1, and the two proteins cooperate in promoting epithelial-type exon usage of select pre-mRNAs. Thus, TGF-β-induced KHSRP silencing is central in a pathway leading to gene-expression changes that contribute to the cellular changes linked toEMT.

Negative autoregulation of BMP dependent transcription by SIN3B splicing reveals a role for RBM39.

BMP signalling is negatively autoregulated by several genes including SMAD6, Noggin and Gremlin, and autoregulators are possible targets for enhancing BMP signalling in disorders such as fibrosis and pulmonary hypertension. To identify novel negative regulators of BMP signalling, we used siRNA screening in mouse C2C12 cells with a BMP-responsive luciferase reporter. Knockdown of several splicing factors increased BMP4-dependent transcription and target gene expression. Knockdown of RBM39 produced the greatest enhancement in BMP activity. Transcriptome-wide RNA sequencing identified a change in Sin3b exon usage after RBM39 knockdown. SIN3B targets histone deacetylases to chromatin to repress transcription. In mouse, Sin3b produces long and short isoforms, with the short isoform lacking the ability to recruit HDACs. BMP4 induced a shift in SIN3B expression to the long isoform, and this change in isoform ratio was prevented by RBM39 knockdown. Knockdown of long isoform SIN3B enhanced BMP4-dependent transcription, whereas knockdown of the short isoform did not. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing, and that RBM39 plays a role in this process.

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

Decreased Usage of Specific Scrib Exons Defines a More Malignant Phenotype of Breast Cancer With Worsened Survival

SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.

IGEMS: an integrated model for identification of alternative exon usage events.

DNA microarrays and RNAseq are complementary methods for studying RNA molecules. Current computational methods to determine alternative exon usage (AEU) using such data require impractical visual inspection and still yield high false-positive rates. Integrated Gene and Exon Model of Splicing (iGEMS) adapts a gene-level residuals model with a gene size adjusted false discovery rate and exon-level analysis to circumvent these limitations. iGEMS was applied to two new DNA microarray datasets, including the high coverage Human Transcriptome Arrays 2.0 and performance was validated using RT-qPCR. First, AEU was studied in adipocytes treated with (n = 9) or without (n = 8) the anti-diabetes drug, rosiglitazone. iGEMS identified 555 genes with AEU, and robust verification by RT-qPCR (∼90%). Second, in a three-way human tissue comparison (muscle, adipose and blood, n = 41) iGEMS identified 4421 genes with at least one AEU event, with excellent RT-qPCR verification (95%, n = 22). Importantly, iGEMS identified a variety of AEU events, including 3′UTR extension, as well as exon inclusion/exclusion impacting on protein kinase and extracellular matrix domains. In conclusion, iGEMS is a robust method for identification of AEU while the variety of exon usage between human tissues is 5–10 times more prevalent than reported by the Genotype-Tissue Expression consortium using RNA sequencing.

Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2′-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.

Abstract A45: Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer

Abstract The information content of the human transcriptome is expanded by the presence of alternative mRNA isoforms produced by alternative transcription initiation, alternative splicing, and alternative polyadenylation. Alternative isoforms can possess distinct oncogenic and tumor suppressive functions by altering gene products and may therefore relate to aggressiveness of disease. A major challenge in prostate cancer biology is the need to distinguish indolent from aggressive disease. Elucidating the molecular determinants of aggressiveness has major implications for developing new genomic diagnostics for clinicians to use, and epigenetic regulation of gene expression that results in more aggressive cancers are compelling drug targets. Here, we hypothesize that differential DNA methylation in gene bodies regulates the production of tumor-promoting alternative transcription events in aggressive prostate cancer. DNA methylation may serve a regulatory role in concert with regulatory proteins and sequence features because transcription start site selection and alternative splicing occur co-transcriptionally. To detect interdependencies between differential DNA methylation and alternative isoforms, we utilized RNA-seq and Illumina HumanMethylation 450K array data produced by The Cancer Genome Atlas for 537 normal prostate and prostate cancer tissue specimens. Reference-guided de novo transcriptome assembly was performed, and fractional usage was computed at both the exon and isoform level for 4,734 expressed genes that contain gene-body differential methylation sites on the 450K array. We identified a set of 120 genes with correlations (|Spearman correlation| &amp;gt; 0.5, FDR adj. p-value &amp;lt; 0.05 and difference &amp;gt; 5% between fractional usage when grouped by the top and bottom quintile of DNA methylation) between the exon or isoform usage fraction and gene body CpG site methylation level. Of these, 53 genes contain differential DNA methylation and exon or isoform usage specific to aggressive disease. This gene set includes SEPT9, which is known to have a cell migration promoting alternative promoter regulated by with DNA methylation changes in breast cancer, as well as two genes with prior links to prostate cancer aggressiveness (MT2A and TDRD1). At the exon level, there were 6 cases where at least one differential methylation site associated with first exon usage, 7 with last exons, and 121 with internal exons. At the isoform level, 92 isoforms that associated with gene body DNA methylation contained alternative promoter usage, 80 contained alternative end usage, 71 contained alternative splice donor/acceptor sites, and 82 contained exon skipping/inclusion events. Additionally, 4 isoforms were predicted to be targets for nonsense mediated decay (NMD) by inducing message frameshifts that reveal internal stop codons. The diversity of this distribution suggests that DNA methylation may interact with a variety of different regulatory mechanisms that occur co-transcriptionally. While the 450K array only covers a small fraction of gene body CpG sites, our analysis supports the possibility of alternative isoform usage regulated by prostate cancer-specific and aggressiveness subtype-specific DNA methylation changes. Future work will interrogate the exact nature of the mechanisms by which DNA methylation changes cross talk with regulatory factors in alternative splicing, promoter usage, and polyadenylation. Such mechanisms may be targetable pharmacologically for tumor suppressive effect. Citation Format: Jeffrey M. Bhasin, Angela H. Ting. Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A45.

Exon-centric regulation of ATM expression is population-dependent and amenable to antisense modification by pseudoexon targeting.

ATM is an important cancer susceptibility gene that encodes a critical apical kinase of the DNA damage response (DDR) pathway. We show that a key nonsense-mediated RNA decay switch exon (NSE) in ATM is repressed by U2AF, PUF60 and hnRNPA1. The NSE activation was haplotype-specific and was most promoted by cytosine at rs609621 in the NSE 3′ splice-site (3′ss), which is predominant in high cancer risk populations. NSE levels were deregulated in leukemias and were influenced by the identity of U2AF35 residue 34. We also identify splice-switching oligonucleotides (SSOs) that exploit competition of adjacent pseudoexons to modulate NSE levels. The U2AF-regulated exon usage in the ATM signalling pathway was centred on the MRN/ATM-CHEK2-CDC25-cdc2/cyclin-B axis and preferentially involved transcripts implicated in cancer-associated gene fusions and chromosomal translocations. These results reveal important links between 3′ss control and ATM-dependent responses to double-strand DNA breaks, demonstrate functional plasticity of intronic variants and illustrate versatility of intronic SSOs that target pseudo-3′ss to modify gene expression.

Characterization of visceral and subcutaneous adipose tissue transcriptome in pregnant women with and without spontaneous labor at term: implication of alternative splicing in the metabolic adaptations of adipose tissue to parturition.

The aim of this study was to determine gene expression and splicing changes associated with parturition and regions (visceral vs. subcutaneous) of the adipose tissue of pregnant women. The transcriptome of visceral and abdominal subcutaneous adipose tissue from pregnant women at term with (n=15) and without (n=25) spontaneous labor was profiled with the Affymetrix GeneChip Human Exon 1.0 ST array. Overall gene expression changes and the differential exon usage rate were compared between patient groups (unpaired analyses) and adipose tissue regions (paired analyses). Selected genes were tested by quantitative reverse transcription-polymerase chain reaction. Four hundred and eighty-two genes were differentially expressed between visceral and subcutaneous fat of pregnant women with spontaneous labor at term (q-value <0.1; fold change >1.5). Biological processes enriched in this comparison included tissue and vasculature development as well as inflammatory and metabolic pathways. Differential splicing was found for 42 genes [q-value <0.1; differences in Finding Isoforms using Robust Multichip Analysis scores >2] between adipose tissue regions of women not in labor. Differential exon usage associated with parturition was found for three genes (LIMS1, HSPA5, and GSTK1) in subcutaneous tissues. We show for the first time evidence of implication of mRNA splicing and processing machinery in the subcutaneous adipose tissue of women in labor compared to those without labor.

Exon- and contraction-dependent functions of titin in sarcomere assembly.

Titin-truncating variants (TTNtvs) are the major cause of dilated cardiomyopathy (DCM); however, allelic heterogeneity (TTNtvs in different exons) results in variable phenotypes, and remains a major hurdle for disease diagnosis and therapy. Here, we generated a panel of ttn mutants in zebrafish. Four single deletion mutants in ttn.2 or ttn.1 resulted in four phenotypes and three double ttn.2/ttn.1 mutants exhibited more severe phenotypes in somites. Protein analysis identified ttnxu071 as a near-null mutant and the other six mutants as hypomorphic alleles. Studies of ttnxu071 uncovered a function of titin in guiding the assembly of nascent myofibrils from premyofibrils. By contrast, sarcomeres were assembled in the hypomorphic ttn mutants but either became susceptible to biomechanical stresses such as contraction or degenerated during development. Further genetic studies indicated that the exon usage hypothesis, but not the toxic peptide or the Cronos hypothesis, could account for these exon-dependent effects. In conclusion, we modeled TTNtv allelic heterogeneity during development and paved the way for future studies to decipher allelic heterogeneity in adult DCM.

Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.

Different Mutant Splicing Factors Cause Distinct Missplicing Events and Give Rise to Different Clinical Phenotypes in Myelodysplastic Syndromes

IntroductionSplicing factor (SF) mutations represent a novel class of driver mutations in myelodysplastic syndromes (MDS), where SF3B1 and SRSF2 are most frequently affected. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets whose abnormal splicing is responsible for the pathogenesis of MDS.MethodsWe enrolled a total of 480 patients with MDS, for whom complete clinical and pathological data were available. RNA sequencing was performed for bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells from 215 MDS patients. Observed splicing junctions were compared between samples with and without each SF mutation. In SF-mutated cases, NMD could cause severe degradation of abnormal transcripts and obscure the effect of SF-mutants. To sensitively detect abnormal transcripts otherwise degraded by nonsense-mediated RNA decay (NMD), analysis was also performed on BM/MNCs from 7 patients and CD34+ bone marrow cells from 3 patients with or without inhibition of NMD by cycloheximide (CHX). Common mutations and copy number variations were also investigated using targeted sequencing.ResultsSF3B1 and SRSF2 mutations were associated with distinct clinical phenotypes and outcomes. SF3B1-mutated cases typically showed isolated erythroid dysplasia and high proportion of ring sideroblasts, whereas SRSF2 mutations correlated with a significantly higher incidence of myeloid and megakaryocyte dysplasia (P<.001), higher proportion of bone marrow blasts (P=.02) and lower degree of erythroid dysplasia and proportion of ring sideroblasts (P<.001). SF3B1- and SRSF2-mutated myeloid neoplasms were also associated with a significantly different outcome, SRSF2-mutated neoplasms having a significantly shorter survival (HR=5.35, P<.001).To explore the molecular basis of these distinct features in terms of splicing defects, RNA sequencing data from SF3B1-mutated (n = 68) and SRSF2-mutated (n = 39) BM/MNCs and CD34+ cells were compared with those without known SF mutations (n = 91) to detect splicing events significantly enriched in SF-mutated cells. In total 748 and 589 splicing events were enriched in SF3B1- and SRSF2-mutated samples. Among these, 203 (27%) of SF3B1-specific events were observed almost exclusively in SF3B1-mutated samples;193 (95%) were caused by misrecognition of 3' splice sites of which ~50% resulted in frameshift. In contrast, in SRSF2-mutated cases, the predominant defects were alternative exon usage, which accounted for for 80% of the abnormalities. However, the effect of mutant-SRSF2 on abnormal splicing was generally small, with 89% showing only <3× more abnormal transcripts in SRSF2-mutated. Similar results were obtained for BM/MNCs for both mutations.Splicing defects of both SF-mutations involved substantially different set of genes. Aberrant splicing enriched in SF3B1- and SRSF2-mutated samples involved 12 and 7 cancer-related genes defined by the Cancer Gene Census with no genes overlapped in between. Of special interest among these was EZH2, which showed 2 SRSF2-associated alternative exons with a premature termination codon. EZH2 transcripts having these exons are expected to be susceptible to NMD-mediated degradation. A similar defect was also detected in another component of the polycomb repressive complex 2 (PRC2), implicated in compromised function of PRC2 in SRSF2-mutated cases. On the other hand, 2 genes involved in mitochondrial heme synthesis were significantly affected in SF3B1-mutated samples. In addition, an additional gene engaged in heme synthesis, ABCB7, was identified from experiments using NMD inhibition to detect 'masked splicing'. ABCB7 is one of the causative genes for congenital sideroblastic anemia and uniformly showed reduced expression in SF3B1-mutated samples, most likely due to abnormal splicing.ConclusionSF3B1 and SRSF2 mutations have distinct impacts on clinical phenotypes and outcomes together with RNA splicing. SF3B1 mutations caused misrecognition of 3' splice sites, frequently resulting in truncated gene products and/or decreased expression due to NMD. SRSF2 mutations were characterized by modest but more widespread alterations in exon usage of genes including multiple components of PRC2. Our results provide insights into the pathogenesis of SF-mutated MDS. DisclosuresNo relevant conflicts of interest to declare.

Ubiquitin Editing of a Spliceosome Auxiliary Factor By TRAF6 Links Chronic TLR Signaling with Hematopoietic Defects and Myelodysplasia

Toll-like receptors (TLR) are known for regulating myeloid homeostasis and response to infection, but chronic activation of TLR pathways can also lead to hematopoietic stem and progenitor cell (HSPC) dysfunction. Furthermore, mutations that lead to constitutive activation of TLR pathways contribute to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS); however, the underlying cellular and molecular mechanisms are unknown. As a means of chronically activating TLR signaling within HSPCs, we generated a mouse model by elevating expression of TRAF6 in hematopoietic cells (Vav-TRAF6). TRAF6 is a downstream TLR-effector with ubiquitin (Ub) E3 ligase activity, and is overexpressed in MDS HSPCs. Vav-TRAF6 mice developed progressive leukopenia and anemia, and exhibited myeloid skewing and dysplasia. Eventually, over half of Vav-TRAF6 mice succumbed to a bone marrow (BM) failure associated with MDS. Despite increased frequencies of immunophenotypic HSPCs in the BM, Vav-TRAF6 HSPCs are functionally defective as evidenced by impaired colony formation and reduced in vivo competitiveness. The hematopoietic phenotype due to TRAF6 overexpression was still manifest upon transplantation, indicating that the effect is hematopoietic cell intrinsic. Consistent with the cellular effects observed with chronic TLR activation, elevated TRAF6 expression results in MDS/BMF by altering intrinsic HSPC properties.Gene expression and exon level analyses revealed that Vav-TRAF6 HSPCs exhibit discrete and durable alterations in RNA splicing patterns. The family of small G-protein GTPases emerged as a relevant pathway whose activity is affected by missplicing of Arhgap1, a GTPase activating protein. Aberrant skipping of Arhgap1 exon 2 resulted in reduced Arhgap1 protein and constitutive Cdc42 GTPase activation. Inhibition of Cdc42 activity with a pharmacological inhibitor partially reversed myeloid-biased differentiation of Vav-TRAF6 HSPCs in vivo, indicating that missplicing of Arhgap1 and increased Cdc42 activity accounts for several HSPC defects.To identify the mechanism underlying TRAF6-induced RNA splicing, we employed a global Ub-enrichment screen for novel TRAF6 substrates, and uncovered hnRNPA1, an RNA-binding protein that regulates exon usage. hnRNPA1 is ubiquitinated by TRAF6 adjacent to and within its first RNA-binding domain. hnRNPA1 binding sites were significantly enriched within misspliced exons in Vav-TRAF6 HSPCs and in primary human AML samples with elevated TRAF6 expression, indicating that TRAF6 overexpression induces exon skipping via hnRNPA1. The requirement of hnRNPA1 in TRAF6-induced exon skipping was confirmed as knockdown of hnRNPA1 significantly reduced Arhgap1 exon 2 skipping in Vav-TRAF6 HSPC. Moreover, depletion of hnRNPA1 reversed Vav-TRAF6 hematopoietic defects in vivo, unequivocally validating the importance of hnRNPA1 in TRAF6-mediated exon skipping and function of HSPCs.Our findings uncover a novel mechanism by which sustained TLR signaling, via TRAF6-mediated ubiquitination of hnRNPA1, alters RNA splicing and contributes to MDS-associated HSPC defects in part by activating Cdc42. These results indicate a novel function for Ub signaling in coordinating transcriptional initiation and alternative splicing by TLR signaling pathway within the immune system and in premalignant hematologic diseases, such as MDS. DisclosuresStarczynowski:Celgene Corporation: Research Funding.

Major advances in physiology: celebrating a centenary of contributions by women. Introduction.

Major advances in physiology: celebrating a centenary of contributions by women. Introduction.

Alternative Splicing Signatures in RNA‐seq Data: Percent Spliced in (PSI)

Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon-centric manner and compared across conditions.

Transcriptome Sequencing to Identify Transcription Factor Regulatory Network and Alternative Splicing in Endothelial Cells Under VEGF Stimulation.

This study aims to investigate the mechanisms underlying the response of human umbilical vein vascular endothelial cells (HUVECs) to vascular endothelial growth factor (VEGF) stimulation. HUVECs were treated with or without 16 ng/mL VEGF for 4 days, and RNA was extracted from HUVECs. After sequencing and data filtering (tool: NGS QC Toolkit), clean data were mapped to genome hg19 (tool: TopHat2). Thereafter, 154 differentially expressed genes (DEGs) were identified between VEGF group and control group (tool: Cuffdiff), and DEGs were enriched in 11 pathways associated with cytokine receptor interaction and chemokine signaling. Protein-protein interaction network of DEGs was constructed (tool: STRING), and ISG15 and MX1 were hub DEGs. The regulatory network of DEGs and transcription factors (TFs) (tool: TRED database) was also constructed, and CCL2 and FN1 (hub DEGs) were co-regulated by NFKB1 and RELA (hub TFs). Moreover, exon usage and alternative splicing were analyzed (tool: DEXSeq), and the splicing of ADORA2A was altered under VEGF stimulation. VEGF might influence HUVECs proliferation and migration, as well as angiogenesis process by regulating the expression of ISG15, MX1, CCL2, FN1, and ADORA2A. However, more research studies are still required to verify these predictions.