Identification of Potential Therapeutic Targets Using Genome-Wide Analysis of Alternative Splicing (AS) In Patients with Acute Myeloid Leukemia (AML)

Abstract Abstract 177 Current therapy cures only a fraction of adults with AML, and it is clear that new treatments, specifically targeted to the genetic or epigenetic lesions that cause the disease, are needed to improve outcome. While monoclonal antibodies against cell surface antigens have been effective therapeutics in other hematologic malignancies, there has only been limited success in AML, partly due to the sharing of many antigens with normal stem cells. We have previously shown that alternative splicing (AS) of genes is common in AML cells when compared to normal CD34+ cells, and here we report efforts to identify alternatively spliced genes encoding surface proteins that could yield novel antibody targets. In order to identify genes that are differentially spliced between AML and normal progenitor cells (NPC), we extended our original genome-wide AS study of 27 AML patient samples to include 123 AML patient samples, 8 NPC's, and 11 AML cell lines. For this study, samples were hybridized to the Affymetrix Human Exon 10ST array. Results were analyzed using commercially available Xray software from Biotique Systems. We identified 217 genes that were differentially spliced in > 35% of patients with AML compared to NPC. An average of 30 differentially spliced genes was observed in each individual patient. Splicing events in any given patients ranged from 10 to 50. Of the 217 commonly spliced genes, 33 genes were found to encode trans-membrane proteins. Three genes, NOTCH2, FLT3 and CD13, were selected for further study. First, the exon array results were validated by RT-PCR, qRT-PCR, and DNA cloning/sequencing from 10 patients. In each case, at least one aberrant splice form was identified that is predicted to encode one or more alternative extracellular regions of the protein. These AS events did not otherwise change the open reading frame of these genes. Two different splice variants of NOTCH2 (NOTCH2-Va and -Vb) were detected in more than 80% (P=0.0001) AML patients, but at undetectable or minimal levels in NPC's. Similarly, we detected three novel aberrant splice variants of FLT3 and CD13, which we have designated as FLT3-Va, -Vb, and -Vc and CD13-Va, -Vb, -Vc, at least one of which was detected in all AML patient samples tested, alone or with full length transcripts. The breakdown for the frequency of expression for these variants in AML patients are as follows: FLT3 -Va (68/123; 55.3%; P=0.001); -Vb (62/123; 50.4%; P=0.003); -Vc (14/123; 11.4%; P=0.15) and CD13-Va (73/123; 59.3%; P=0.001); -Vb (57/123; 46.3%; P=0.005); -Vc (34/123; 27.6%; P=0.042). None of these variants were observed in BM or PB samples from normal donors. We evaluated the expression frequency of these splice variants in patient groups with different FAB subtypes, as well as in patients groups with cytogenetically normal or complex AML. This analysis identified frequent expression of NOTCH2-Va splice variant in patients (more than 85% patients) with M0, M1, M2, M5 and M6 AML, while FLT3-Va and -Vb variants were detected in more than 50 % of patients diagnosed with M1, M2 and M5 AML. CD13-Va and -Vb variants were identified in more than 60 % of M6 AML, while CD13-Va is expressed more than 80% of M1 AML. Interestingly, the NOTCH2-Va splice variant is expressed in nearly 90% of patients with cytogenetically complex AML, while NOTCH2-Va, FLT3-Va, -Vb and FLT3-Va were expressed more than 60% of patients with normal karyotype AML. Moderate expression frequency of other splice variants has been observed in AML patients with different subgroups analyzed. Overall, our results from genome-wide AS analysis suggest that alternative splicing is a common event in AML, with some splice variants being detected in a significant number of different subgroups of patients. The clinical consequences and significance of this finding, as well as frequency of novel variant transcripts in large population of AML is currently the focus of further investigation. Screening and correlation analysis of additional 300 patients samples obtained at time of diagnosis as well as during relapse are in progress. Certain of the more common splice variants may generate new targets for the development of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.

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Genome-Wide Aberrant Splicing in Patients with Acute Myeloid Leukemia (AML) Indetifies Potential Novel Targets

Blood ◽

10.1182/blood.v118.21.761.761 ◽

2011 ◽

Vol 118 (21) ◽

pp. 761-761

Author(s):

Sophia Adamia ◽

Michal Bar-Natan ◽

Benjamin Haibe-Kains ◽

Hervé Avet-Loiseau ◽

Laurence Lode ◽

...

Keyword(s):

Splice Variant ◽

Splice Variants ◽

Test Group ◽

Myeloid Cell ◽

Surface Proteins ◽

Sequencing Analysis ◽

Aberrant Splicing ◽

Genome Wide ◽

Cd34 Cells ◽

Antigenic Epitopes

Abstract Abstract 761 Background: AML is characterized by specific chromosomal translocations along with a number of other recurrent mutations. However, it is also becoming clear that AML cells have frequent epigenetic abnormalities that also contribute to transformation. Alternative splicing (AS) is an epigenetic event that is used in normal cells to generate protein diversity from single genes. Using genome-wide screening approaches, we have shown that alternative splicing of numerous genes is aberrant in most cases of AML, and that several common myeloid cell surface proteins may have novel and potentially antigenic epitopes as a result. Methods: Using Affymetrix Human Exon 10ST arrays we performed a genome-wide analysis of AS in bone marrow (BM) aspirates from two cohorts (training cohort including 40 patients and test cohort including 41 patients) of patients with AML (total 81 patients) and CD34+ BM progenitor cells from 8 normal donors (NDs). After principle component analysis, 6 patients were removed from the test group, because they were outliers. In final array analysis for the test group we analysed 35 patients. Aberrant splicing of several selected genes was confirmed through cloning and sequencing analysis followed evaluating expression patterns of cloned novel splice variant transcripts in 193 patients compared to NDs. To evaluate the clinical significance of AS in AML we determined if there were any correlations between novel aberrant splice variant expression and clinical features of patients. Results and conclusions: We found that AS is a common event in AML involving many genes in any given patient. Overall, we identified ∼550 genes that were significantly spliced in patients compared to normal CD34+ cells. Three genes encoding myeloid cell surface proteins, NOTCH2, CD13, and FLT3, were selected for further study. By cloning and sequencing analysis we found that novel splice variants of the selected genes are the results of either exon skipping, usage of cryptic 5' or 3' splice sites on exons and/or partial retention of an intron. We also found that these splice variants encode proteins, as assessed by expressing splice variant transcripts in the HEK293 cell line. To identify the most frequently expressed variants of NOTCH2, FLT3 and CD13 in AML, we performed expression profiling of these transcripts and their variants in an independent validation group of 193 AML patients. These analyses identified NOTCH2-Va (74%), FLT3-Va (50%) and CD13-Va (60%) as the most frequently expressed variants in AML. Expression of these novel splice variants, other than NOTCH2-Vb, was undetectable in BM CD34+ cells, normal monocytes or neutrophils. To examine whether expression of novel variants are associated with disease evolution, we performed longitudinal monitoring of expression levels of NOTCH2-FL, FLT3-FL, and CD13-FL and their splice variants in paired samples of 13 patients taken over the course of the disease. Our study suggests the “splicing profile” normalizes in remission and recurs when patients relapse. Correlation analysis among NOTCH2, CD13 variants and FLT3 splice variants, and clinical features of patients with AML, showed that splice variant NOTCH2-Va expression is significantly negatively associated with overall survival (OS) of patients (P=0.008). Our study also showed that deregulated expression of NOTCH2-Va identifies two subgroups of patients within the intermediate-risk profile patients. We also observed significantly inferior OS of the patients that overexpressed NOTCH2-Va within this group (P=0.007). Thus, NOTCH2-Va expression can be used to stratify patients with cytogenetically determined intermediate-risk profiles. Overall, our study demonstrates that genome-wide study of AS can be used to discover new prognostic markers, and potentially creates new surface antigenic epitopes that could be targeted in AML. Disclosures: No relevant conflicts of interest to declare.

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Characterization of three alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g74130

Botany ◽

10.1139/cjb-2013-0173 ◽

2013 ◽

Vol 91 (12) ◽

pp. 840-849 ◽

Cited By ~ 4

Author(s):

Joshua Powles ◽

Katharine Sedivy-Haley ◽

Eric Chapman ◽

Kenton Ko

Keyword(s):

Alternative Splicing ◽

Alternative Splice ◽

Splice Variant ◽

Serine Proteases ◽

Transmembrane Domain ◽

Splice Variants ◽

Genes Encoding ◽

Different Characteristics ◽

Alternative Splice Variants

Rhomboid serine proteases are grouped into three main types — secretases, presenilin-like associated rhomboid-like (PARL) proteases, and “inactive” rhomboid proteins. Although the three rhomboid groups are distinct, the different types are likely to operate within the same cell or compartment, such as observed in the plastids of Arabidopsis. There are four distinct plastid rhomboid genes at play in Arabidopsis plastids, two for active types (At1g25290 and At5g25752) and two for inactive forms (At1g74130 and At1g74140). The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290. To understand how the plastid rhomboid system works, it is necessary to identify all rhomboid forms in play. To this end, this study was designed to examine the alternative splicing activities of At1g74130, one of the two genes encoding proteolytically “inactive” plastid rhomboids. The exon mapping and DNA sequencing results obtained here indicate the presence of three prominent alternative splice variants in the At1g74130 transcript population. The dominant splice variant, L, encodes the full-length protein. The other two splice variants, M and S, produce proteins lacking sections from the carboxyl transmembrane domain region. The splice variants M and S appear to be at levels with functional potential and appear to adjust relative to each other during development and in response to changes in the level of Tic40, a component of the plastid translocon. The splice variant proteins themselves exhibit different characteristics with respect to rhomboid protein–substrate interactions. These differences were observed in bacterial co-expression pull-down assays and in yeast mitochondrial studies. When considered together, the data suggest that the alternative splicing of At1g74130 bears functional significance in Arabidopsis and is likely to be part of a mechanism for diversifying plastid rhomboid function.

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Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53

Genome ◽

10.1139/g07-074 ◽

2007 ◽

Vol 50 (10) ◽

pp. 946-953 ◽

Cited By ~ 15

Author(s):

Kim Disher ◽

Adonis Skandalis

Keyword(s):

Oxidative Stress ◽

Alternative Splicing ◽

Molecular Mechanisms ◽

Splice Variants ◽

Dna Lesions ◽

Mrna Splicing ◽

Aberrant Splice ◽

Biological Phenomenon ◽

Mrna Splice ◽

Human Genes

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.

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Splice variants’ role in mediating different disease states in tissue requiring ion transduction through calcium channel for function

10.7287/peerj.preprints.2717v1 ◽

2017 ◽

Author(s):

Wenfa Ng

Keyword(s):

Alternative Splicing ◽

Calcium Channel ◽

Alternative Splice ◽

Splice Variant ◽

Splice Variants ◽

Energetic Cost ◽

Hypoxic Stress ◽

Tissue Samples ◽

Hek 293 ◽

Selection Of

Structure informs function, and this may be the evolutionary reason why alternative splicing, which is capable of generating different variants of the same protein, arise. But, given the energetic cost of generating different splice variants for testing their capability at a specific task, which incurs cellular functional uncertainty; as well as the exertion of differing physiological effects on cells that may translate into diseased states, what is the evolutionary advantage of this process? Additionally, what are the factors that select a specific variant for a presented task? Using heart tissue samples exposed to hypoxia stress as model system, this research idea entails the illumination of single nucleotide polymorphisms (SNP) of the calcium channel transporter, Cav 1.2 gene in the population through gene sequencing followed by bioinformatic analysis for alternative splice sites. This would be followed by a scan for alternative splice variants through colony polymerase chain reaction using universal primers for Cav 1.2 gene. Confirmation of splice variant identity through Western blot laid the stage for subsequent efforts at cloning and expressing the variant gene in HEK 293 cells lacking endogenous expression of Cav 1.2, for biophysical characterization of calcium conduction through patch clamp electrophysiology. In parallel, structural elucidation efforts necessitate the purification of the calcium channel via hydrophobic interaction or reversed phase liquid chromatography after its heterologous expression in a bacterial host. But, biophysical and biochemical characterization does not speak of the signaling and metabolic pathways laying the path to generation of the splice variant(s). Hence, discovery approaches such as RNA-seq and mass spectrometry proteomics could uncover the molecular mysteries at the transcript and protein level that help guide the selection of specific splice variant in response to hypoxic stress, where HIF is a candidate pathway. Implementing this approach from the retrospective angle of examining diseased human tissue samples provide one important facet for uncovering possible mechanisms driving the generation of a splice variant. However, the complementary prospective approach of identifying the molecular basis and processes for responding to hypoxia in a cell line such as HEK 293 would help provide confirmatory evidence in understanding the key drivers of physiological response to lack of oxygen at the cellular level. Collectively, this research route would illuminate both the nucleotide informational basis of alternative splicing in calcium channel Cav 1.2 as well as identify the molecular mechanisms enabling the selection of specific splice variants useful for conferring, at the cell and tissue level, ability to withstand hypoxic stress without significant negative effects on cell function. Interested readers can expand on the ideas presented.

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Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

10.1101/2021.02.18.431627 ◽

2021 ◽

Author(s):

Veerle Lemmens ◽

Bart Thevelein ◽

Svenja Kankowski ◽

Hideaki Mizuno ◽

Jochen C. Meier ◽

...

Keyword(s):

Chronic Pain ◽

Single Molecule ◽

Splice Variant ◽

Single Channel ◽

Pain Treatment ◽

Conflicts Of Interest ◽

Splice Variants ◽

Formal Analysis ◽

Glycine Receptor ◽

Link Type

AbstractGlycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson’s and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the α3K splice variant. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR α3 channels, which is important to understand and tackle α3 related diseases.SummaryThe glycine receptor α3 is key to the central nervous system’s physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize α3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis.DeclarationsFundingWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF).Conflicts of interest / competing interestsNo conflicts of interest apply.Ethics approvalNot applicableAvailability of data and materialAll data and material are available upon request.Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS athttp://www.cup.uni-muenchen.de/pc/lamb/software/pam.htmlor hosted in Git repositories underhttp://www.gitlab.com/PAM-PIE/PAMandhttp://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available athttp://pam.readthedocs.io.Author contributionsConceptualization Meier J.C., Brône B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

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Analysis of splice variants of the human protein disulfide isomerase (P4HB) gene

BMC Genomics ◽

10.1186/s12864-020-07164-y ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Daniela Kajihara ◽

Chung-Chau Hon ◽

Aimi Naim Abdullah ◽

João Wosniak ◽

Ana Iochabel S. Moretti ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Alternative Splicing ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Splice Variant ◽

Splice Variants ◽

Muscle Cells ◽

Protein Disulfide

Abstract Background Protein Disulfide Isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily with crucial roles in endoplasmic reticulum proteostasis, implicated in many diseases. The family prototype PDIA1 is also involved in vascular redox cell signaling. PDIA1 is coded by the P4HB gene. While forced changes in P4HB gene expression promote physiological effects, little is known about endogenous P4HB gene regulation and, in particular, gene modulation by alternative splicing. This study addressed the P4HB splice variant landscape. Results Ten protein coding sequences (Ensembl) of the P4HB gene originating from alternative splicing were characterized. Structural features suggest that except for P4HB-021, other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. These indicated widespread expression but significant variability in the degree of isoform expression among distinct tissues and even among distinct locations of the same cell, e.g., vascular smooth muscle cells from different origins. P4HB-02, P4HB-027 and P4HB-021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. Expression of such variants was validated by qRT-PCR in some cell types. The most consistently expressed splice variant was P4HB-021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation. Conclusions Our study details the splice variant landscape of the P4HB gene, indicating their potential role to diversify the functional reach of this crucial gene. P4HB-021 splice variant deserves further investigation in vascular smooth muscle cells.

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Alternative splicing of the androgen receptor in polycystic ovary syndrome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418216112 ◽

2015 ◽

Vol 112 (15) ◽

pp. 4743-4748 ◽

Cited By ~ 44

Author(s):

Fangfang Wang ◽

Jiexue Pan ◽

Ye Liu ◽

Qing Meng ◽

Pingping Lv ◽

...

Keyword(s):

Androgen Receptor ◽

Alternative Splicing ◽

Polycystic Ovary Syndrome ◽

Splice Variants ◽

Polycystic Ovary ◽

Endocrine Disorders ◽

Ovary Syndrome ◽

Insertion And Deletion ◽

Expression Of Genes ◽

Genome Wide

Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ∼62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS.

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Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls

Reproduction ◽

10.1530/rep-13-0343 ◽

2014 ◽

Vol 147 (2) ◽

pp. 241-252 ◽

Cited By ~ 23

Author(s):

F Guo ◽

B Yang ◽

Z H Ju ◽

X G Wang ◽

C Qi ◽

...

Keyword(s):

Alternative Splicing ◽

Sperm Motility ◽

Splice Variants ◽

Sperm Quality ◽

Methylation Pattern ◽

Luciferase Reporter ◽

Aberrant Splice ◽

Sequencing Analysis ◽

Exonic Splice ◽

Holstein Bulls

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of theSPEF2gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that theSPEF2was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis.SPEF2-SV1was differentially expressed in the sperms of high-performance and low-performance adult bulls;SPEF2-SV2presents the highest expression in testis and epididymis;SPEF2-SV3was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 ofSPEF2, located within a putative exonic splice enhancer, potentially producedSPEF2-SV3and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated toSPEF2methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation ofSPEF2expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

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Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0210109 ◽

1998 ◽

Vol 21 (2) ◽

pp. 109-120 ◽

Cited By ~ 17

Author(s):

L Bresson-Bepoldin ◽

MC Jacquot ◽

W Schlegel ◽

SR Rawlings

Keyword(s):

Alternative Splicing ◽

Adenylate Cyclase ◽

Single Cell ◽

Splice Variant ◽

Cell Biology ◽

Splice Variants ◽

Pituitary Cells ◽

Rt Pcr ◽

Pituitary Adenylate Cyclase

Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology.

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Genome-Wide Analysis of Alternative Splicing Points to Novel Leukemia Relevant Genes in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2391.2391 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2391-2391

Author(s):

Anna Dolnik ◽

Andreas Gerhardinger ◽

Ursula Botzenhardt ◽

Sabrina Heinrich ◽

Richard Schlenk ◽

...

Keyword(s):

Alternative Splicing ◽

Myeloid Leukemia ◽

Splice Variants ◽

Cell Cycle Control ◽

Rt Pcr ◽

Genome Wide ◽

Alternatively Spliced ◽

Splice Forms ◽

Exon Microarray ◽

Acute Myeloid

Abstract Abstract 2391 Poster Board II-368 Alternative mRNA splicing represents an effective mechanism of regulating gene function as well as a key element to increase the coding capacity of the human genome. Today, an increasing number of reports illustrates that aberrant splicing events can contribute to human disease and that alterations in the splicing machinery are common and functionally important for cancer development. Aberrant splice forms can for example have genome-wide effects by deregulating key signaling pathways. However, for most of the aberrant mRNA transcripts detected it remains unclear whether they directly contribute to the malignant phenotype or just represent a by-product of cellular transformation. Thus, more comprehensive analyses of the transcriptome splicing are warranted in order to get novel insights into the biology underlying malignancies like, e.g., acute myeloid leukemia (AML). Here, we performed a genome-wide screening of splicing events in AML using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). We analyzed forty AML cases with complex karyotypes and twenty Core Binding Factor (CBF) AML cases (entered on a multicenter trial for patients <60 years, AMLSG 07-04) using this microarray approach allowing the detection of splice variants. In order to detect alternative splicing events distinguishing different leukemia subgroups we applied a commercial and an open source software tool: XRAY version 3.9 (Biotique Systems) and the OneChannelGUI package for R (version 1.10.7 available at http://www.bioinformatica.unito.it/oneChannelGUI/). Using XRAY supervised analysis comparing subgroups of CBF and complex karyotype AML we identified 1120 transcripts to be potentially alternatively spliced. In parallel, the analysis of the same AML subgroups using the OneChannelGUI package in R revealed 1439 candidates with an overlap of only 211 genes. Of these transcripts, that have been indicated by both programs as potentially alternatively spliced, selected candidates were further investigated by RT-PCR, quantitative RT-PCR and sequence analysis for the presence of splice-variants. Of 26 candidate genes studied, we could confirm alternative splice forms for 5 genes that might potentially be involved in driving leukemogenesis, such as the protein coding gene arginine methyltransferase 1 (PRMT1), which regulates transcription through histone methylation and participates in DNA damage response. Furthermore, we could confirm differential exon usage in the protein tyrosine phosphatase non-receptor type (PTPN6) transcript, which encodes for a negative regulator of numerous signaling pathways involved in cell cycle control and apoptosis. Similarly, the mRNA of the protein Rho GTPase activating protein 4 (ARHGAP4), which has been shown to regulate cell motility, was alternatively spliced between CBF and complex karyotype subgroups. In summary, these first gene expression data demonstrate that the attempt to elucidate the splicing of transcriptome in AML by applying Exon microarray technology is challenging in particular with regard to the currently available software solutions. Nevertheless, our results show that this approach offers the ability to detect novel alternatively spliced candidate genes. Being involved in cell cycle control, regulation of transcription or remodeling of the cytoskeleton, alternative splicing of these genes might play a potential role in the pathomechanism of distinct AML subgroups. Thus, in the future more extensive Exon array profiling with more sophisticated software solutions at hand is likely to provide additional insights into the molecular mechanisms of leukemogenesis and might reveal novel targets for refined therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.

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