V2 Transcript Research Articles

Abstract 1396: Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer

Abstract Background: Among all breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prognosis, in part attributable to the fact that TNBC lacks targets for effective molecularly-targeted therapies. The concept that EGFR inhibitor drugs could be used as a targeted treatment against TNBC has been put forth because roughly 50% of TNBC express high levels of EGFR. However clinical trials targeting EGFR did not significantly improve patient outcomes. Our recent work offers a potential explanation as to why EGFR inhibitors failed and supports an innovative therapeutic approach combining an EGFR tyrosine kinase inhibitor with the novel antioxidant biologic, CAT-SKL. Our data indicate that inhibition of cancer stem cell-like cells depends on antioxidant-induced downregulation of an alternative mRNA splice variant of the methyl-CpG binding domain 2 gene, MBD2_v2. Objective and Methods: The purpose of the present study was to evaluate the importance of MBD2_v2 in TNBC and better understand how CAT-SKL regulates its expression. Using TNBC cell lines we investigated the effects of MBD2_v2 expression on tumor initiation capacity. Furthermore we studied MBD2_v2 mRNA levels in an in vivo tumor xenograph mouse model of TNBC and in human TNBC samples using an existing database. Then by RNA-seq analysis we assessed how CAT-SKL regulates MBD2_v2 expression in TNBC cells through differential gene expression. Results: The results support that MBD2_v2 expression is regulated by redox signaling in TNBC cells, which is tightly linked to TNBC cell tumor initiation capacity. Furthermore, MBD2_v2 was upregulated in tumors harvested from overweight mice, which also displayed increased tumor take rate, suggesting that a pro-inflammatory tumor microenvironment could play a role in promoting MBD2_v2 expression. In addition, RNA-seq analysis identified an overrepresentation of mRNA splicing factors with expression downregulated by CAT-SKL treatment of TNBC cells. From this gene set two splicing co-factors are highlighted: SRRM1 and CPSF3. These co-factors are critical for splicing factor function, 3’ end formation and polyadenylation; and both have been linked via protein-protein interaction to a relevant exonic splicing enhancer site in exon 3 of the MBD2 _v2 transcript. Finally, according to the Kaplan Meier Plotter database high MBD2_v2 levels in BC were associated with shorter relapse free survival. Conclusion: Our investigation sheds light on the relevance of MBD2_v2 expression in TNBC, and how a select antioxidant regulates its expression. The application of which holds promise of a novel, targeted therapeutic modality for TNBC, the subtype with the worst prognosis and highest need for treatment options. Citation Format: Emily A. Girsch, Bin Bao, Cristina Mitrea, Gregory Dyson, Julie Boerner, Lisa Polin, Stan R. Terlecky, Aliccia Bollig-Fischer. Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2017-1396

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.

Abstract 1930: The unknown piece of the pie: Molecular markers in triple-negative lung cancer.

Abstract Lung cancer is the main cause of cancer mortality worldwide. Approximately ∼80% of lung cancer cases are non-small cell lung cancer (NSCLC) in type and >50% of NSCLC are adenocarcinoma in histopathology. A shifting paradigm in the field of pulmonary oncology is the identification of molecular markers that are therapeutic targets and/or provide prognostic information. The recent recognition of the role biomarkers such as EGFR, KRAS, and ALK play in NSCLC adenocarcinoma patients demonstrates this trend. Although these three highly characterized molecular markers make up one-quarter to two-thirds of NSCLC adenocarcinoma patients, the remaining population, termed “Triple-Negative Lung Cancer” (TNLC), has yet to be fully defined. The complete absence of therapeutic targets for triple-negative lung cancer patients undeniably supports the need to identify up-regulated and mutated genes that better define this poor prognostic cohort. Recent genome-wide expression profiles subdivided TNLC patients into a poor prognostic group based on the overexpression of the DEP domain containing 1 gene (DEPDC1) (Okayama et al., 2011). We hypothesized that overexpression of DEPDC1 correlates with a specific sub-population of adenocarcinoma patients. Our initial studies indicate that DEPDC1 is constitutively expressed in both normal lung as well as lung cancer specimens. DEPDC1 transcripts are present as two variants (V1 and V2), with constitutive expression of the V1 variant in normal lung. We hypothesized that expression of the V2 transcript could be unique to NSCLC adenocarcinomas. Screening of FFPE NSCLC adenocarcinoma specimens using a proprietary qPCR approach demonstrated the presence of the DEPDC1 V2 variant but at levels that were not sufficient to serve as a sole biomarker. We therefore investigated whether the ratio of DEPDC1 V2 expression relative to V1 could serve as a biomarker in TNLC. Our preliminary data suggest that the ratio of V2 to V1 expression of DEPDC1 does in fact segregate TNLC into specific sub-populations. These studies have exciting diagnostic as well as therapeutic implications as Phase I/II studies using novel peptide vaccines derived against DEPDC1 have proven tolerable and efficacious for patients with bladder cancer and could potentially be extended to target these newly identified TNLC sub-populations. Citation Format: Brock L. Schweitzer, Kasey D. Lawrence, John Handshoe, Rachel Skelton, Lindsay Chatfield, Liquan Xue, Stephan W. Morris, David R. Hout. The unknown piece of the pie: Molecular markers in triple-negative lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1930. doi:10.1158/1538-7445.AM2013-1930

A Comprehensive Transcriptome Assembly of Pigeonpea (Cajanus cajan L.) using Sanger and Second-Generation Sequencing Platforms

A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ∼8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.

Highly Ordered Spatial Organization of the Structural Long Noncoding NEAT1 RNAs within Paraspeckle Nuclear Bodies

Paraspeckles (PSPs) are nuclear bodies associated with the retention in the nucleus of specific mRNAs. Two isoforms of a long noncoding RNA (NEAT1_v1/Menε and NEAT1_v2/Menβ) are required for the integrity of PSPs. Here, we analyzed the molecular organization of PSPs by immuno- and in situ hybridization electron microscopy. Detection of the paraspeckle markers PSPC1 and P54NRB/NONO confirm the identity between PSPs and the previously described interchromatin granule-associated zones (IGAZs). High-resolution in situ hybridization of NEAT1 transcripts revealed a highly ordered organization of IGAZ/PSPs. Although the 3.7-kb NEAT1_v1 and the identical 5' end of the 22.7-kb NEAT1_v2 transcripts are confined to the periphery, central sequences of NEAT1_v2 are found within the electron-dense core of the bodies. Moreover, the 3' end of NEAT1_v2 also localize to the periphery, indicating possible architectures for IGAZ/PSPs. These results further suggest that the organization of NEAT1 transcripts constrains the geometry of these bodies. Accordingly, we observed in HeLa and NIH 3T3 cells that IGAZ/PSPs are elongated structures with a well-defined diameter. Our results provide new insight on the ability of noncoding RNAs to form subcellular structures.

Overexpression of human glycolipid transfer protein transcript (GLTP), but not novel splice variants GLTP_v1 or GLTP_v2, induces apoptosis

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer and serves as the conformational prototype defining the conserved GLTP superfamily. In humans, the single‐copy GLTP gene at 12q24.11 encodes the five‐exon GLTP transcript. Herein, two previously unknown GLTP splice variants are identified and functionally evaluated. Exon1 was the only exon common to the GLTP, GLTP_v1, and GLTP_v2 transcripts. In GLTP_v1, a new exon has been discovered within sequence previously designated GLTP intron2. The GLTP splice variants are highly tissue specific compared to the more ubiquitous GLTP transcript. Immunolocalization reveals distribution of exogenously‐expressed GLTP_i1 and GLTP_i2 throughout HeLa cells and, unlike GLTP, presence within nuclei. Exogenous GLTP expression induces S phase cell cycle arrest and apoptosis in HeLa cells. The pro‐apoptotic effect of GLTP occurs independently of glycolipid binding/transfer. In contrast, exogenous expression of GLTP_v1 and GLTP_v2 protects cells from apoptosis induced by hydrogen peroxide or etoposide without altering the cell cycle. Neither GLTP splice variant affects the expression or glycolipid transfer activity of GLTP. The data show for the first time that overexpression of GLTP, but not GLTP_v1 or GLTPv2, induces cell apoptosis, underscoring the biological diversity of the GLTP gene [Support: NIH GM45928, CA121493 & Hormel Fnd.]

Identification of multiple vasotocin receptor cDNAs in teleost fish: Sequences, phylogenetic analysis, sites of expression, and regulation in the hypothalamus and gill in response to hyperosmotic challenge

Vasopressin and its homolog vasotocin regulate hydromineral balance, stress responses, and social behaviors in vertebrates. In mammals, the functions of vasopressin are mediated via three classes of membrane-bound receptors: V1a-type, V1b-type and V2-type. To date, however, only a single class of vasotocin receptor has been identified in teleost fish. Here, cDNAs encoding three putative vasotocin receptors – two distinct V1a-type receptor paralogs ( V1a1 and V1a2) and a previously undescribed V2-type receptor ( V2) – and a single isotocin receptor were isolated and sequenced from the Amargosa pupfish ( Cyprinodon nevadensis amargosae). RT-PCR revealed that mRNAs for these receptors differed in expression patterns with V1a1 mRNAs abundant in the brain, pituitary and testis, V1a2 transcripts at greatest levels in brain, heart and muscle, V2 transcripts most common in the gills, heart and kidney, and isotocin receptor mRNAs abundant in the midbrain, pituitary and gonads. In response to an acute hyperosmotic challenge, pro-vasotocin and V2 mRNA levels in the hypothalamus decreased, while transcripts of V1a1 in the hypothalamus and V1a2 in the gills increased. Partial transcripts for structurally related V2-type, as well as multiple V1a-type, receptors were also identified in other teleosts, suggesting that multiple vasotocin receptors may be present in many Actinopterygii fishes.

Human endothelial and platelet septin SEPT11: Cloning of novel variants and characterisation of interaction partners

Septins are cytoskeletal GTPases forming heteropolymeric complexes involved in processes characterised by active membrane movement such as cytokinesis, vesicle trafficking, and exocytosis. Septins are expressed in non-mitotic cells such as neurons and platelets. SEPT11 belongs to the SEPT6 group and was identified as interaction partner of SEPT5. We cloned and characterised novel SEPT11 variants and investigated interaction partners of SEPT11 in platelets and human umbilical vein endothelial cells. An endothelial cell library was used for cloning novel SEPT11 variants. Using Northern analysis the different SEPT11 transcripts were illustrated. Interaction studies were performed using yeast two-hybrid system, precipitation, FRET, and immunofluorescence microscopy. We demonstrate that SEPT11 partners with SEPT2, SEPT4 and SEPT7 using yeast two-hybrid system and precipitation. The interaction of SEPT11 with SEPT7 is also demonstrated by FRET. In addition to the known SEPT11 transcript (SEPT11_v1) we identified a novel SEPT11 variant (SEPT11_v2) as interaction partner of SEPT4 and SEPT7. Library screening of an endothelial cell library also revealed the presence of this novel SEPT11_v2 transcript. In addition, a third SEPT11 variant (SEPT11_v3) was identified. Expression of SEPT11_v1 and of SEPT11_v2 and SEPT11_v3 in human brain regions was investigated by Northern analysis. Further interaction partners of SEPT11 are characterised using immunofluorescence. Co-localisation of SEPT2, SEPT4, SEPT7 and SEPT11 with tubulin and transferrin receptor (endocytotic marker) is demonstrated. In addition, co-localisation of SEPT4 and SEPT11 with the vesicle-associated protein synaptobrevin 1 (VAMP1), but not clearly with actin, was shown. Only SEPT2 and SEPT7 definitely co-localised with actin, but not clearly with VAMP1.

Transcriptional Regulation of the Human Growth Hormone Receptor (hGHR) Gene V2 Promoter by Transcriptional Activators and Repressor

The V2 transcript is the major ubiquitously expressed human GH receptor (hGHR) mRNA in all tissues examined to date. In a previous investigation, we defined the V2 promoter as TATA-less and exhibiting many characteristics of a housekeeping gene promoter. We also demonstrated that its basal activity is determined by several different cis-regulatory regions within both the promoter and the V2 exon. In the present study, we used luciferase-reporter, site-directed mutagenesis, gel shift, chromatin immunoprecipitation, and quantitative RT-PCR assays to investigate the ability of certain transcription factors to regulate hGHR V2 transcription through these regions in mammalian cells, including human adipocytes. Ets1 was found to transactivate the V2 proximal promoter through specific Ets sites. Two CCAAT/enhancer-binding protein (C/EBP) family members [C/EBP-homologous protein (CHOP) and C/EBPbeta] enhanced V2 transcription via different pathways: indirectly, by association with a V2 exon region (CHOP), and directly, using a V2 proximal promoter noncanonical binding site (C/EBPbeta). The Notch signaling mediator, Hes1, potently suppressed V2 promoter activity through interaction with two Hes sites within the V2 exon. We propose that these transcriptional factors regulate hGHR V2 expression by acting as downstream nuclear effectors, linking specific signaling cascades (e.g. MAPK and Notch) triggered by different growth factor-, development-, and nutrition- as well as stress-related stimuli. Our data also suggest that these factors are likely to be important in the differentiation-induced increase in V2 mRNA expression in adipocytes, with Ets1 and CHOP functioning at the preadipocyte stage to prepare the cells for differentiation and increasing C/EBPs and decreasing Hes1 levels contributing during adipocyte maturation.

Structure and Activity of the Human Growth Hormone Receptor (hGHR) Gene V2 Promoter

Human GH (hGH) has important effects on growth as well as carbohydrate, fat, and protein metabolism. These actions require the presence of normal levels of a functional hGH receptor (hGHR) on the surface of target cells. hGHR gene expression is characterized by the use of several 5'-noncoding exons and alternative splicing, resulting in the generation of multiple mRNA isoforms. The hGHR V2 transcript is predominant in most tissues, including human fat. However, factors regulating its ubiquitous expression have remained unidentified. The present study was aimed at characterizing the mechanisms regulating hGHR V2 transcription. Two major V2 transcriptional start sites were identified by primer extension assays. The V2 proximal promoter is TATA-less, with several characteristics of a housekeeping gene promoter. Transient transfection analyses of 2.6 kb of the 5'-flanking region of V2 confirmed its promoter activity in multiple primate cell lines. Similar promoter activity patterns were observed in human SGBS preadipocytes and mature adipocytes but with much higher V2 promoter activity in mature adipocytes, suggesting that changes in the availability of specific factors during adipocyte differentiation play a role in V2 promoter regulation. Serial deletion and mutation analyses revealed that transcription of hGHR V2 in different cell types, including adipocytes, is determined by a core promoter and distinct inhibitory and activation domains in the 5'-promoter region as well as within the V2 exon. Our data suggest that V2 transcription is the result of a complex interplay involving multiple factors, to ensure appropriate expression of hGHR in different hGH target cells.

Characterization of Growth Hormone Receptor Messenger Ribonucleic Acid Variants in Human Adipocytes

Human GH exerts profound effects on adiposity through its specific receptor, hGHR. Eight hGHR mRNAs are produced by the hGHR gene due to splicing from alternate 5'-untranslated region first exons into a common acceptor site upstream of the start codon in exon 2. Four transcripts (V2, V3, V5, V9) are ubiquitously expressed, whereas the other four (V1, V4, V7, V8) are expressed only in normal postnatal liver, suggesting that different promoter usage is a mechanism for developmental- and tissue-specific regulation of the hGHR gene. Because it is unknown whether this occurs in adipocytes, we screened human adipocyte cDNA for hGHR mRNAs using 5'-rapid amplification of cDNA ends. Eighty-nine percent of the clones were V2-like, 3% were V3-like, and 8% were five new mRNA variants (VA-VE). All new 5'-untranslated region sequences mapped within the hGHR 5'flanking region. RT-PCR assays showed expression in multiple fetal and adult tissues, and, thus, they are not adipocyte specific. We compared expression of hGHR mRNAs in adult liver, adult fat, and the human preadipocyte SGBS cell line, using duplex RT-PCR. In liver, V1 and V2 are the major hGHR mRNAs, whereas in adipose, V2 predominates; VA and VC are expressed at similar lower levels in both. In SGBS preadipocytes, approximately 70% of hGHR mRNA is V2. During differentiation, total hGHR and V2 transcripts are markedly up-regulated [hGHR: 2.3 +/- 0.2-fold (mean +/- se), P < 0.01; V2: 3.0 +/- 0.8, P < 0.03], whereas other variants also increased but remained relatively minor transcripts. We have identified five new hGHR mRNA variants. Because the V2 transcript is predominant in adipocytes at all developmental stages, the mechanisms regulating its expression should be examined.

Alternative Splicing Forms of the Human CD1D Gene in Mononuclear Cells

CD1d is a critical molecule for the presentation of lipid antigens to natural killer (NK) T cells. To investigate the molecular complexity of CD1d, alternatively spliced transcripts in peripheral blood mononuclear cells from three healthy subjects were analyzed by PCR and sequencing methods. We found eight alternatively spliced variants of the CD1D gene (V1–V8), seven of which are newly established variants (V2–V8). V1 and V4 are in-frame; however, the other six variants (V2, V3, V5–V8) are out-of-frame. V1, V2, V4, and V5 lack a β2-microglobulin binding site (α3 domain), indicating the unstable presentation of the CD1d molecule on the surface. In V2 and V5, the transmembrane region is absent, supporting a soluble CD1d. In the V3–V8 variants, the antigen binding region (α1 and α2 domains) is partially defective, suggesting incomplete functional products. In contrast, the V1 and V2 transcripts bear the complete antigen binding site, resulting in functional proteins. Especially, the V2 splicing variant might function as an inhibitory soluble CD1d molecule and regulate the presentation of antigens on APC to NKT cells.

Differential and temporal expression of the vitellogenin genes in Drosophila grimshawi.

The three vitellogenin transcripts from Drosophila grimshawi have an average size of 1,600 nucleotides as determined using denaturing electrophoretic conditions. Southern analysis showed that the large quantity of vitellogenin mRNAs in adult female fat body cells is not a reflection of specific gene amplification. The quantitative differences in mRNA accumulation between fat body and follicle cells, which are in concert with the onset of their translation, indicate that vitellogenin synthesis entails the regulated expression of individual genes. The expression of the vitellogenin genes during follicle development is stage specific: V1 and V2 expression starts at late stage 7, while V3 is delayed by one stage. Maximum transcription of all three genes occurs at stage 10 whereas at stage 12 none of the transcripts is present. These results suggest that, either there is more than one regulatory signal, or there is one to which each gene reacts differently. Surprisingly, in male fat body cells a V2 transcript has been detected which is also present in the poly(A)+RNA fraction: the function and the purpose of this particular vitellogenin mRNA in male fat body cells are unknown. Neither of the other two vitellogenin transcripts have been detected in male fat body cells.