Exon Array Analysis Research Articles

JAK2 Dependent GM-CSF Signaling Triggers SRSF2 Phosphorylation: A Potential Cause Of Mutation-Independent RNA Splicing Aberrancy In CMML

SRSF2 is a RNA-binding protein frequently mutated in CMML tasked with marking exon-intron boundaries during alternative splicing. SRSF2 contains a domain consisting of serine-arginine repeats that are dynamically phosphorylated and comprise a major regulatory mechanism in RNA capture. It has been previously demonstrated that EGFR signaling can elicit the phosphorylation of SRSF2 in an AKT dependent manner, resulting in functional alterations in exon usage of SRSF2 target genes. We previously reported that GM-CSF hypersensitivity is a reproducible feature of CMML. Because AKT is a known signaling intermediate of GM-CSF, we hypothesize that aberrant GM-CSF signaling could induce mutation-independent splicing abnormalities in CMML via phosphorylation of SRSF2. To test this hypothesis, we selected CMML samples that displayed GM-CSF hypersensitivity but lacked mutations by Sanger sequencing in 5 splicing-related genes commonly mutated in myeloid malignancies (SRSF2, SF3B1,U2AF1, ZRSF2,and PRPF40B) and labeled this group as CMML ‘splicing WT'. A second sample set included CMML specimens with isolated SRSF2 mutation (SRSF2 ‘splicing MT'). Bone marrow mononuclear cells from the SRSF2 ‘splicing WT' group (n=4), CMML ‘splicing MT' group (n=4), and normal controls (n=4) were lymphodepleted (CD3 and CD19) to enrich for the clonal myeloid population. RNA was extracted and whole transcriptome RNA sequencing and exon arrays were performed to compare the relative global exon usage of CMML ‘splicing WT', CMML ‘splicing MT', and normal controls. Briefly, the human exon 1.0 ST array was used for exon array analysis and splice index was calculated with the Exon/Gene Array Analyzer Web Tool (Gellert Bioinformatics 2009). RNA sequencing data was generated and aligned with TopHat2 (Kim Genome Bio 2013) while read counts and exon usage assignments were made with DexSeq (Anders Genome Res 2012). Splice indices determined by exon array demonstrated statistically significant exon usage differences between CMML ‘splicing MT' and normal controls consistent with previously published results (Figure 1a). Interestingly, when CMML ‘splicing WT' was compared to normal controls, a statistically significant difference in exon usage was also identified (Figure 1b). Further, when ‘splicing MT' and ‘splicing WT' were compared by splice index, differences in exon usage differences were dramatically reduced indicating similar splicing abnormalities in the CMML 'splicing WT' group (1c). Euclidian hierarchical clustering at the gene- and exon-level separated normal controls from CMML groups but failed to separate ‘splicing MT' from ‘splicing WT', supporting the notion that mutation-independent splicing aberrancy may characterize CMML (1d). Global patterns in exon usage were also tested by whole transcriptome RNA-sequencing and were similar to those described above identified by exon array splice index. [Display omitted] We first attempted to determine if the transcript levels of core splicing components in CMML ‘splicing WT' were altered but were unable to identify differences in expression when compared to CMML 'splicing MT' and controls. Next we explored the possibility that GM-CSF signaling, known to be upregulated in CMML, could post-translationally modify SRSF2. The THP-1 monocytic leukemia cell line was stimulated with 10ng/ml of GM-CSF for 15 minutes. Cell lysates were immediately prepared and western blot analysis was used to assess SRSF2 phosphorylation after stimulation with GM-CSF, demonstrating induction of pSRSF2 at 15 minutes as accompanied by induction of the known GM-CSF signaling intermediates pJAK2, pAKT, and pERK. Pharmacologic inhibition of these intermediates abrogated GM-CSF-induced pSRSF2, whereas only the JAK 1/2 inhibitor ruxolitinib was able to inhibit pSTAT5, pAKT, and pERK, suggesting that JAK2 is a proximal kinase in the signaling cascade. Our results indicate that splicing abnormalities are present in CMML specimens lacking mutations in splicing-related genes. GM-CSF induces phosphorylation of SRSF2 for post-translational regulation of the RS domain. Low dose GM-CSF dependent pSRSF2 and functional splicing analyses are currently underway in primary CMML ‘SRSF2 WT' samples to directly determine whether GM-CSF hypersensitivity is responsible for mutation-independent splicing aberrancies. Disclosures:List: Celgene: Membership on an entity's Board of Directors or advisory committees.

Identification of key signaling molecules downstream of cAMP that regulate insulin secretion

In the pancreatic islet, the unique heterotrimeric G protein α‐subunit, Gαz, inhibits adenylate cyclase, reducing cyclic adenosine monophosphate (cAMP) levels. cAMP is an important potentiator of glucose‐stimulated insulin secretion (GSIS) and Gαz‐null islets have enhanced GSIS. To further delineate the mechanisms downstream of cAMP that impact GSIS, an exon array analysis was performed in islets isolated from 10‐week‐old male Gαz‐null and control mice. Candidate genes were flagged as being differentially expressed in Gαz‐null islets. To further test the mechanism for gene regulation, highly glucose‐and cAMP‐responsive Ins‐1 832/3 insulinoma cells were treated with various stable cAMP analogs. PGC‐1α was induced greater than 4‐fold with increasing 8‐CPT‐cAMP dose and 8‐fold with increasing 8‐CPT‐cAMP treatment time. 8‐CPT‐cAMP activates both the Epac and PKA pathways. Treatment with an Epac‐specific cAMP analog, 8‐CPT‐2′‐O‐Me‐cAMP, had no effect on PGC‐1α suggesting PKA as the specific activator downstream of cAMP. Thus, PGC‐1α may play an important role in cAMP dependent insulin secretion and further functional characterization of PGC‐1α in the β‐cell may help explain the phenotype of increased GSIS in Gαz‐null islets. This work was funded by JDRF grant 17–2011‐608 (to M.E.K).

Exon Array Analysis of Alternative Splicing of Genes in SOD1G93A Transgenic Mice

Alternative splicing is a common strategy for creating functional diversities of proteins. While conventional identification of splice variants generally targets individual genes in amyotrophic lateral sclerosis, we present a novel exon-centric array that allows genome-wide identification of splice variants and concurrently provides analysis of gene expression. Compare 1 was asymptomatic SOD1G93A transgenic mice with nontransgenic littermates; compare 2 was symptomatic with asymptomatic transgenic mice. RT-PCR was performed to validate. Pathway and GO analysis were performed on abnormal genes. These findings could guide us to demonstrated the potential influence of mutant human CuZn-SOD1 and of splicing regulation in pathological processes.

Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

BackgroundFor decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described.ResultsFrom the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties.Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation.ConclusionsAn Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene expression studies strongly suggested that the difference in leaf Cd accumulation between the two specific tobacco cultivars is dependent solely on genetic factors and genetic variability rather than on the environment.

Alternative Splicing and Expression of Class II Tubulin Beta (TUBB2B) Are Associated with Outcome in Diffuse Large B-Cell Lymphoma

Abstract 1557 Background:The aim was to compare differentially expressed exons and splicing variants between diffuse large B-cell lymphoma (DLBCL) patients, who had relapsed or remained in remission after dose dense chemoimmunotherapy. Design and methods:We performed genome-wide exon array analysis from four DLBCL cell lines and 38 tumor tissues from young (<65 years) DLBCL patients with high-risk (aaIPI>1) disease. The patients were treated in a Nordic phase II protocol with six courses of R-CHOEP-14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and high dose cytarabine. At the time of the analysis, median follow up was 34 months, predicted 3-year progression free survival (PFS) 78% and overall survival (OS) 79%. RNA for quantitative PCR validation was available from 20 patients. Two DLBCL cell lines and eight patient samples were further analyzed with high throughput RNA sequencing. In addition, microarray data set from 233 DLBCL patients treated with chemoimmunotherapy (Lymphoma/Leukemia Molecular Profiling Project (LLMPP)) was utilized for validation. Results:Differentially expressed exons between relapsed and non-relapsed patients were screened using criteria of p ≤ 0.05 and fold change ≥1.6 converting to 566 differentially expressed genes, of which 131 coded proteins. One of the identified genes with possible alternative splicing was TUBB2B, which encodes therapeutic target of taxanes and vinca alkaloids. The expression of TUBB2B, and specifically the expression of exon 3, was found to be suppressed in relapsed patients in comparison to patients remaining in remission. Differential expression of TUBB2B whole transcript and exon 3 was confirmed with RNAseq and quantitative PCR. Studies in lymphoma cell lines provided further support for the existence of different TUBB2B isoforms. According to Kaplan Meier estimates the patients with high (>median) expression levels of TUBB2B exon 3 had better 3-year PFS and lymphoma related OS rates than the patients with low expression levels (95% vs. 61%, p=0.015 for PFS, 100% vs. 75%, p=0.024 for OS). The prognostic significance of TUBB2B gene expression was validated in LLMPP data set (3-year OS 80% vs. 67%, p=0.040). Conclusions:The results provide evidence that differential expression and splicing of TUBB2B gene can discriminate the outcome of homogenously treated high risk DLBCL patients. Disclosures:No relevant conflicts of interest to declare.

Abstract IA06: Identification of tumor-associated cassette exons in human cancer through exon-array and RNA-Seq data analysis.

Abstract Many evidences report that alternative splicing, the mechanism producing mRNAs and proteins with different structures and functions from the same gene, is altered in cancer cells. Thus, the identification and characterization of cancer-specific splice variants may give large impulse to the discovery of novel diagnostic and prognostic tumor biomarkers, as well as of new targets for more selective and effective therapies. We developed novel and effective computational tools for performing a genome-wide analysis of the alternative splicing pattern of human genes using both exon array and RNA-Seq data. Notably, we sought to identify “cassette exon” generated by exon skipping differentially expressed in the tumor and normal conditions. To this aim we analyzed data obtained by exon array hybridization from clear cell renal carcinoma (ccRC) and colorectal cancer (CRC) samples as well as from the corresponding normal controls. Furthermore, high-throughput transcriptome sequencing from CRC and control samples were also analyzed. By using our computational strategy we identified several cassette exons differentially expressed in the tumoral and normal conditions. In order to test the effectiveness of our computational strategy, we experimentally verified the occurrence of tumor- and normal-specific cassette exons by RT-qPCR assay. Remarkably, we were able to confirm “in vivo” the differential expression of these predicted cassette exons in patients affected by CRC and ccRC. This study presents a novel computational methodology for the identification of tumor-associated transcript variants to be used as cancer molecular biomarkers, provides its experimental validation, and reports specific candidate biomarkers for CRC and ccRC. Citation Format: Alessio Valletti, Orazio Palumbo, Mattia D'Antonio, Tiziana Castrignano, Margherita Gigante, Massimo Carella, Ernesto Picardi, Anna Maria D'Erchia, Elena Ranieri, Graziano Pesole. Identification of tumor-associated cassette exons in human cancer through exon-array and RNA-Seq data analysis. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA06.

A method to identify RNA A-to-I editing targets using I-specific cleavage and exon array analysis

RNA A-to-I editing is the most common single-base editing in the animal kingdom. Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Mouse knockout models deficient in ADAR activities show lethal phenotypes associated with defects in nervous system, failure of hematopoiesis and reduced tolerance to stress. While several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of ADARs remain unknown. Here we report a method to systematically analyze RNA A-to-I editing targets by combining I-specific cleavage and exon array analysis. Our results show that I-specific cleavage on editing sites causes more than twofold signal reductions in edited exons of known targets such as Gria2, Htr2c, Gabra3 and Cyfip2 in mice. This method provides an experimental approach for genome-wide analysis of RNA A-to-I editing targets with exon-level resolution. We believe this method will help expedite inquiry into the roles of RNA A-to-I editing in various biological processes and diseases.

Alteration of POLDIP3 Splicing Associated with Loss of Function of TDP-43 in Tissues Affected with ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by selective loss of motor neurons. In the ALS motor neurons, TAR DNA-binding protein of 43 kDa (TDP-43) is dislocated from the nucleus to cytoplasm and forms inclusions, suggesting that loss of a nuclear function of TDP-43 may underlie the pathogenesis of ALS. TDP-43 functions in RNA metabolism include regulation of transcription, mRNA stability, and alternative splicing of pre-mRNA. However, a function of TDP-43 in tissue affected with ALS has not been elucidated. We sought to identify the molecular indicators reflecting on a TDP-43 function. Using exon array analysis, we observed a remarkable alteration of splicing in the polymerase delta interacting protein 3 (POLDIP3) as a result of the depletion of TDP-43 expression in two types of cultured cells. In the cells treated with TDP-43 siRNA, wild-type POLDIP3 (variant-1) decreased and POLDIP3 lacking exon 3 (variant-2) increased. The RNA binding ability of TDP-43 was necessary for inclusion of POLDIP3 exon 3. Moreover, we found an increment of POLDIP3 variant-2 mRNA in motor cortex, spinal cord and spinal motor neurons collected by laser capture microdissection with ALS. Our results suggest a loss of TDP-43 function in tissues affected with ALS, supporting the hypothesis that a loss of function of TDP-43 underlies the pathogenesis of ALS.

Erratum: Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

Erratum: Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

Leucine‐rich repeat kinase 2 and alternative splicing in Parkinson's disease

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD) and are associated with pleiomorphic neuropathology. We hypothesize that LRRK2 mediates its pathogenic effect through alternative splicing of neurodegeneration genes. Methods used in this study included western blotting analysis of subcellular protein fractions, exon-array analysis of RNA from cultured neuroblastoma cells transfected with LRRK2 expression vectors, and reverse-transcription polymerase chain reaction (RT-PCR) of RNA from cultured cells and postmortem tissue. Overexpression of the LRRK2 G2019S mutant resulted in a significant (2.6-fold; P = 0.020) decrease in nuclear transactive response DNA-binding protein 43 levels. Exon-array analyses revealed that wild-type LRRK2 had a significant effect on the expression of genes with nuclear (P < 10(-22) ) and cell-cycle functions (P < 10(-15) ). We replicated changes in gene expression in 30% of selected genes by quantitative RT-PCR. Overexpression of LRRK2 resulted in the altered splicing of two genes associated with PD, with an increased inclusion of exon 10 of microtubule-associated protein tau (1.7-fold; P = 0.001) and exon 5 of the alpha-synuclein (SNCA) gene (1.6-fold; P =0.005). Moreover, overexpression of LRRK2 (G2019S) and two mutant genes associated with neurodegeneration, TARDBP (M337V) and FUS (R521H), were associated with decreased inclusion out of the dystonin (DST) 1e precursor exons in SK-N-MC cells. Altered splicing of SNCA (1.9-fold; P < 0.001) and DST genes (log(2) 2.3-fold; P = 0.005) was observed in a cohort of PD, compared with neurologically healthy, brains. This suggests that aberrant RNA metabolism is an important contributor to idiopathic PD.

Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an RNA gain-of-function disorder in which abnormally expanded CTG repeats of DMPK sequestrate a splicing trans-factor MBNL1 and upregulate another splicing trans-factor CUGBP1. To identify a diverse array of aberrantly spliced genes, we performed the exon array analysis of DM1 muscles. We analyzed 72 exons by RT-PCR and found that 27 were aberrantly spliced, whereas 45 were not. Among these, 25 were novel and especially splicing aberrations of LDB3 exon 4 and TTN exon 45 were unique to DM1. Retrospective analysis revealed that four parameters efficiently detect aberrantly spliced exons: (i) the signal intensity is high; (ii) the ratio of probe sets with reliable signal intensities (that is, detection above background P-value=0.000) is high within a gene; (iii) the splice index (SI) is high; and (iv) SI is deviated from SIs of the other exons that can be estimated by calculating the deviation value (DV). Application of the four parameters gave rise to a sensitivity of 77.8% and a specificity of 95.6% in our data set. We propose that calculation of DV, which is unique to our analysis, is of particular importance in analyzing the exon array data.

Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

Characterization of hepatocyte‐specific SRSF1 (SF2/ASF) knockout mice

Alternative splicing is widely believed to have a major impact on almost all biological processes since it increases proteome complexity and thereby controls protein function. SRSF1 (SF2/ASF) is a member of the SR protein family, which are essential for constitutive splicing and fulfilling regulatory functions in alternative splicing. Previously, we have shown that SRSF1 binds to the alternatively spliced exon 11 of the insulin receptor (INSR) and promotes its incorporation in hepatoma cells. To demonstrate the physiological importance of alternative splicing to the regulation of metabolism, we created a hepatocyte‐specific knockout of SRSF1 (SRSF1LKO) by crossing SRSF1 flox/flox mice with albumin‐cre mice, which specifically express the cre recombinase in adult hepatocytes. Mice are born in the expected mendelian ratio and do not show any growth retardation unlike the hepatocytes specific SRSF3 knockout that we have previously characterized. We will present data showing efficient tissue specific deletion of the SRSF1 allele, the reduction in SRSF1 expression, and alterations in INSR splicing. We will also present data on the metabolic and physiological parameters and liver morphology between SFSR1LKO and littermate control mice, as well as alterations in global gene expression and splicing by exon array analysis. SFSR1LKO is an important model to study alternative splicing in vivo.Supported by a Merit Review Award from the Department of Veterans Affairs.

NMD‐deficient Upf3b‐null mice display behavioral and neuropathological defects

To address the physiological role of nonsense‐mediated RNA decay (NMD), we generated mice with a null mutation in the Upf3b NMD gene. While these Upf3b‐null mice are viable, fertile, and grossly normal, a battery of behavioral tests revealed they exhibit a specific defect in sensorimotor gating. This defect is commonly displayed in Schizophrenia patients, a subset of whom have recently been shown by several groups to have mutations in the human UPF3B gene. Neuropathological examination of the frontal cortex, the site responsible for sensorimotor gating, revealed decreased dendritic spine density and mature dendritic spines in pyramidal cells. Intriguingly, such defects are also observed in schizophrenic and autistic patients. To understand the underlying mechanisms by which UPF3B acts in this circuit, we performed exon‐array analysis of the frontal cortex of Upf3b‐null mice. This revealed dysregulation of several mRNAs transcribed from genes mutated in intellectual disability patients, most of which encode proteins that function in sensory perception and cognition. NMD is likely to have a direct role in regulating many of these transcripts, as most have “NMD‐inducing features.” We propose that Upf3b‐null mice are a useful model system to understand the underlying mechanism of sensorimotor gating and how it is disrupted in Schizophrenia patients.This study was supported by the NIH.

Identification of RET gene fusion by exon array analyses in “pan-negative” lung cancer from never smokers

Identification of RET gene fusion by exon array analyses in “pan-negative” lung cancer from never smokers

Curcumin Enhances Neurogenesis and Cognition in Aged Rats: Implications for Transcriptional Interactions Related to Growth and Synaptic Plasticity

BackgroundCurcumin has been demonstrated to have many neuroprotective properties, including improvement of cognition in humans and neurogenesis in animals, yet the mechanism of such effects remains unclear.MethodologyWe assessed behavioural performance and hippocampal cell proliferation in aged rats after 6- and 12-week curcumin-fortified diets. Curcumin enhanced non-spatial and spatial memory, as well as dentate gyrate cell proliferation as compared to control diet rats. We also investigated underlying mechanistic pathways that might link curcumin treatment to increased cognition and neurogenesis via exon array analysis of cortical and hippocampal mRNA transcription. The results revealed a transcriptional network interaction of genes involved in neurotransmission, neuronal development, signal transduction, and metabolism in response to the curcumin treatment.ConclusionsThe results suggest a neurogenesis- and cognition-enhancing potential of prolonged curcumin treatment in aged rats, which may be due to its diverse effects on genes related to growth and plasticity.

Myocardial Alternative RNA Splicing and Gene Expression Profiling in Early Stage Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome.

Mediator Complex Regulates Alternative mRNA Processing via the MED23 Subunit

Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its MED23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit, MED23, but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally, MED23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events, as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that MED23 can regulate hnRNP L occupancy at their coregulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.

10 RNA from FFPE tissue is suitable for exon array analysis if extracted utilizing a robust protocol which incorporates laser capture microdissection

10 RNA from FFPE tissue is suitable for exon array analysis if extracted utilizing a robust protocol which incorporates laser capture microdissection

Alternative splicing of apoptosis-related genes in imatinib-treated K562 cells identified by exon array analysis

Imatinib is the therapeutic standard for newly diagnosed patients with chronic myeloid leukemia (CML). In these patients, imatinib has been shown to induce an apoptotic response specifically in cells expressing the oncogenic fusion protein BCR-ABL. Previous studies in our lab revealed that imatinib-induced apoptosis in K562 cells involves a shift in production of Bcl-x splice isoforms towards the pro-apoptotic Bcl-xS splice variant. Here, we report the findings from our subsequent study to identify other apoptosis-related genes that are differentially spliced in response to imatinib treatment. Gene expression profiling of imatinib-treated K562 cells was performed by the Affymetrix GeneChip® Human Exon 1.0 ST array, and differences in exon-level expression and alternative splicing were analyzed using the easyExon software. Detailed analysis by reverse transcription-PCR (RT-PCR) and sequencing of key genes confirmed the experimental results of the exon array. Our results suggest that imatinib treatment of K562 cells causes a transcriptional shift towards alternative splicing in a large number of apoptotic genes. The present study provides insight into the molecular character of apoptotic leukemia cells and may help to improve the mechanism of imatinib therapy in patients with CML.