Expression Of Alternative Splice Variants Research Articles

Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein.

Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides—besides transcription—an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP—together with the RBM4 splicing factor—in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.

Alternatively spliced BobCAL transcripts alter curd morphotypes in a collection of Chinese cauliflower accessions

The curd of cauliflower (Brassica oleracea L. var. botrytis) is a modified inflorescence that is consumed as a vegetable. Curd formation is proposed to be due to a mutation in the BobCAULIFLOWER (BobCAL) gene, but the genetic relationship between BobCAL variation and curd morphotypes remains obscure. To address this question, we collected and classified a collection of 78 cauliflower accessions into four subpopulations according to curd surface features: smooth, coarse, granular, and hairy curd morphotypes. Through the cDNA sequencing of BobCAL alleles, we showed that smooth and coarse accessions characterized by inflorescence meristem arrest presented a strong association with the 451T SNP (BobCAL_T), whereas granular and hairy accessions marked with floral organ arrest presented an association with 451G (BobCAL_G). Interestingly, all BobCAL alleles were alternatively spliced, resulting in a total of four alternative splice (AS) variants due to the retention of the fourth and/or seventh introns. Among accessions with BobCAL_G alleles, the total expression of all these AS variants in granular plants was almost equal to that in hairy plants; however, the expression of the individual AS variants encoding intact proteins relative to those encoding truncated proteins differed. Hairy accessions showed relatively high expression of the individual variants encoding intact proteins, whereas granular accessions displayed relatively low expression. In smooth cauliflower, the overexpression of the BobCAL_Ga variant caused an alteration in the curd morphotype from smooth to hairy, concurrent with an increase in the expression levels of downstream floral identity genes. These results reveal that alternative splicing of BobCAL transcripts is involved in the determination of cauliflower curd morphotypes.

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF.

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

Aberrant expression of alternative splicing variants in multiple sclerosis - A systematic review.

Alternative splicing is an important form of RNA processing that affects nearly all human genes. The differential expression of specific transcript and protein isoforms holds the potential of novel biomarkers for complex diseases. In this systematic review, we compiled the existing literature on aberrant alternative splicing events in multiple sclerosis (MS). A systematic literature search in the PubMed database was carried out and supplemented by screening the reference lists of the identified articles. We selected only MS-related original research studies which compared the levels of different isoforms of human protein-coding genes. A narrative synthesis of the research findings was conducted. Additionally, we performed a case-control analysis using high-density transcriptome microarray data to reevaluate the genes that were examined in the reviewed studies. A total of 160 records were screened. Of those, 36 studies from the last two decades were included. Most commonly, peripheral blood samples were analyzed (32 studies), and PCR-based techniques were usually employed (27 studies) for measuring the expression of selected genes. Two studies used an exploratory genome-wide approach. Overall, 27 alternatively spliced genes were investigated. Nine of these genes appeared in at least two studies (CD40, CFLAR, FOXP3, IFNAR2, IL7R, MOG, PTPRC, SP140 and TNFRSF1A). The microarray data analysis confirmed differential alternative pre-mRNA splicing for 19 genes. An altered RNA processing of genes mediating immune signaling pathways has been repeatedly implicated in MS. The analysis of individual exon-level expression patterns is stimulated by the advancement of transcriptome profiling technologies. In particular, the examination of genes encoded in MS-associated genetic regions may provide important insights into the pathogenesis of the disease and help to identify new biomarkers.

Functional effects of blocking VEGF/VEGFR2 signaling in the rat urinary bladder in acute and chronic CYP-induced cystitis.

High expression of VEGF is associated with immature angiogenesis within the urinary bladder wall and bladder afferent nerve sensitization, leading to visceral hyperalgesia and pelvic pain. Research suggests a shift in VEGF alternative splice variant (VEGF-Axxxa and VEGF-Axxxb) expression with several pathologies (e.g., neuropathic pain and inflammation) as well as differing effects on pain. Translational studies have also demonstrated increased total VEGF expression in the bladders of women with interstitial cystitis/bladder pain syndrome. In the present study, we quantified VEGF alternative splice variant expression in lower urinary tract tissues under control conditions and with cyclophosphamide (CYP)-induced cystitis. Using conscious cystometry and intravesical instillation of a potent and selective VEGF receptor 2 (VEGFR2) tyrosine kinase inhibitor (Ki-8751, 1 mg/kg) in Wistar rats (male and female) with acute and chronic CYP-induced cystitis and control (no CYP) rats, we further determined the functional effects of VEGFR2 blockade on bladder function. With VEGFR2 blockade, bladder capacity increased (P ≤ 0.01) in male and female control rats as well as in male and female rats with acute (P ≤ 0.05) or chronic (P ≤ 0.01 or P ≤ 0.05, respectively) CYP-induced cystitis. Void volume also increased in female control rats (P ≤ 0.01) and female rats with acute (P ≤ 0.05) or chronic (P ≤ 0.05) CYP-induced cystitis as well as in male control rats (P ≤ 0.05) and male rats with chronic CYP-induced cystitis (P ≤ 0.01). These data suggest that VEGF may be a biomarker for interstitial cystitis/bladder pain syndrome and that targeting VEGF/VEGFR2 signaling may be an effective treatment.

Identification of a Novel TCF4 Isoform in the Human Corneal Endothelium

Alternative splice isoforms of TCF4, a gene implicated in Fuchs corneal dystrophy, have been identified in multiple human tissues outside of the eye. The aim of this study was to identify the transcriptional profile of TCF4 in the corneal endothelium. We extracted RNA from the donor corneal endothelium and performed rapid amplification of cDNA ends. We tested the expression pattern of 1 newly identified isoform (7b) in a panel of cDNA derived from multiple human tissues and included cDNA from corneal endothelial (CE) and retinal pigment epithelial cell lines. To further delineate differential expression of TCF4 splice variants that span CTG18.1, we analyzed expression of 6 alternative splice isoforms that are transcribed from either exon 2 or 3 in RNA extracted from the corneal endothelium of 3 normal donors and a CE cell line. We identified 11 different isoforms in control CE tissue, including 1 isoform (7b) not reported previously. This isoform is enriched specifically in the corneal endothelium and placenta compared with other tissues in a panel of human cDNA. We demonstrate the complex expression profile of TCF4 in the human corneal endothelium and reveal expression of alternative splice variants of TCF4.

Differential Expression of Alternative Splicing Variants in Individuals with Relapsing-Remitting Multiple Sclerosis (P2.382)

Differential Expression of Alternative Splicing Variants in Individuals with Relapsing-Remitting Multiple Sclerosis (P2.382)

Single‐walled carbon nanotubes affect the expression of the CCND2 gene in human U87 glioma cells

The effect of single‐walled carbon nanotubes (SWCNTs) on the expression level of several genes, related to cell cycle control, cell proliferation and migration in human glioma cell line U87 was investigated. The exposure of human U87 glioma cells to single‐walled carbon nanotubes in different concentrations during 24 of 48 h affects the expression of CCND2 (Cyclin D2), DTNA (Dystrobrevin alpha), PARVB (parvin beta), DUSP1 (dual specificity phosphatase 1), PFKFB3 (6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase‐3) and PFKFB4 genes at mRNA level, as well as the expression of alternative splice variants of DTNA, PFKFB3 and PFKFB4 transcripts. In case of CCND2 gene a strong, time and dose‐dependent suppressive effect was observed. The effect of single‐walled carbon nanotubes on the expression level of PFKFB3, PFKFB4, and PARVB was also suppressive but less prominent. At the same time, the expression of DUSP1 and DTNA genes is up‐regulated in cells treated by single‐walled carbon nanotubes. Thus, single‐walled carbon nanotubes exert anti‐proliferative properties in U87 glioma cells, as they down‐regulate the expression of proliferation‐related genes (CCND2, PARVB, PFKFB3, and PFKFB4) and up‐regulate DUSP1 and DTNA genes, which control apoptosis.

Refinement of the spectra of exon usage by combined effects of extracellular stimulus and intracellular factors

Finely tuned differential expression of alternative splice variants contributes to important physiological processes such as the fine-tuning of electrical firing or hearing frequencies; yet the underlying molecular basis for the expression control is not clear. The inclusion levels of four depolarization-regulated alternative exons were measured by RT-PCR in GH3 pituitary cells under different conditions of stimulation and/or RNA interference of splicing factors. The usage of the exons was reduced by membrane depolarization to various extents and was differentially modulated by the knock-down of splicing factors hnRNP L, L-like, I (PTBP1) or K or their combinations. A spectrum of each exon's level was produced under six knock-down conditions and was significantly shifted by depolarization. When all these conditions were considered together, a more refined or expanded spectrum of exon usage was obtained for each of the four exons. As a proof of principle for the molecular basis of the fine-tuning of exon usage, we show in the cases of hnRNP L and LL that their differential effects through the same element or different combinations of RNA sequences by the same factor hnRNP L are critical. The results thus demonstrate that the combined effect of varying extracellular stimuli and intracellular factors/RNA sequences refines or expands the spectra of endogenous exon usage, likely contributing to the fine-tuning of cellular properties.

Differential expression of alternative splice variants of CTLA4 in Kuwaiti autoimmune disease patients

Cytotoxic T lymphocyte associated antigen4 (CTLA4) is a candidate susceptibility gene for the study of autoimmune diseases. The present study sought to explore the expression profile of the CTLA4 gene in autoimmune patients, such as rheumatoid arthritis (RA), systemic lupus erythematous (SLE) and Hashimoto's thyroiditis (HT), compared to healthy controls (HCs). A total of 88 (22 RA, 22 SLE 22 HT, 22 HCs) age-, gender- and ethnicity-matched individuals were recruited. The hypersensitive capillary electrophoresis method was employed to detect the CTLA4 splice variants. PCRs of the patient's cDNA using CTLA4-specific primers followed by cloning and sequencing were used to distinguish the various splice variants. The biochemical properties of all known CTLA4 variants were analysed using the ExPASy and ESEfinder programmes. Six alternatively spliced variants of the CTLA4 gene were detected in this study. These included mCTLA4-672, sCTLA4-562, N-CTLA4-292, L-CTLA4-277, ssCTLA4-214 and K-CTLA4-142bp. K-CTLA4-142bp and N-CTLA4-292bp represented two novel splice variants of the CTLA4 gene. A reduction in the frequency of mCTLA4-672bp and sCTLA4-562bp was observed in SLE and RA patients compared to healthy controls. The shortest splice variant, K-CTLA4-142bp, was predominantly detected in all of the tested cohorts, while the decreased expression of the N-CTLA4-292bp variant was observed in the autoimmune subjects. The exonic splice enhancer motifs of the SRp40 protein were found exactly at the splice junction of wCTLA4 (-ACAGAGC-, 2.7) and K-CTLA4 (-TGAAAAG-, 3.37), and that of the SRp55 protein was found at the splice junction of L-CTLA4 (-TGTGTG-, 2.82). Our study highlights the discrepancies in the expression spectrum of the CTLA4 gene in autoimmune patients and healthy subjects. The abnormal expression pattern of the CTLA4 gene in autoimmune patients suggests that in addition to allelic variation, the expression pattern of CTLA4 could contribute to autoimmunity.

Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na+/K+ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.

Methamphetamine inhibits voltage-gated potassium currents in NG108-15 cells: Possible contribution of large-conductance calcium-activated potassium channels

Methamphetamine (MA), a highly abused amphetamine-like psychostimulant, has surged in popularity worldwide in the last decade. Repeated MA exposure has been shown to affect the alternative splice variant expression of large conductance Ca2+-activated K+ (BK) channels. It remains unclear whether MA affects BK channel activity. The present study investigated the effects of MA on BK channels in NG108-15 mouse neuroblastoma×rat glioma hybrid cells using whole-cell and cell-attached patch clamp techniques. In whole-cell recordings, the macroscopic K+ outward currents were inhibited by MA with an EC50 of 146μM, but not affected by dopamine (DA). It implies that DA is not involved in the effects of MA on K+ outward currents. In cell-attached patches, MA significantly decreased BK channel activity. Moreover, MA significantly decreased the BK channel opener NS1619-evoked whole-cell K+ outward currents and BK channel activity. Finally, the effect of MA on membrane potential was examined by current-clamp configuration. MA caused membrane depolarization and application of NS1619 returned the depolarized potential to resting value. These findings suggest that MA might act as an inhibitor of BK channels, and thereby increase the neuronal excitability and enhance neurotransmitter release.

Protein Kinase C δ (PKCδ) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis: IDENTIFICATION OF PKCδII INHIBITOR

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity.

Abstract 3686: Single cell assays of heterogeneity and clonality in leukemia

Abstract A range of hypotheses have emerged as to why imatinib (a targeted inhibitor of bcr-abl) seems unable to eliminate the most primitive chronic myeloid leukemia (CML) cells, such as bcr-abl amplification and clonal evolution. The biology of the CML imatinib-resistant cell is of critical importance to understanding why despite high percentages of initial clinical responses to imatinib (>80% of patients in chronic phase at some point obtaining complete cytogenetic remission), resistance, relapse and/or progression occurs in 20-30% of cases and most patients in remission still have residual disease detected by sensitive PCR monitoring of the CML specific bcr-abl transcripts. In acute myeloid leukemia (AML) as well, patients are typically treated with a chemotherapeutic regimen that leads to clinical responses in 80-95% of patients. However, over half of these patients will relapse and eventually die of disease suggesting that a subpopulation of leukemic cells are capable of both surviving treatment and causing regrowth of the tumor. In both diseases, it has not been definitively established what the identity of the cells responsible for resistance and relapse is, nor what combination of mutations, gene expression changes, and alternative splicing events are required for these cells’ resistant phenotype. Assessing changes in gene expression in the bulk samples provides a valuable overall picture of the phenotype of the predominant cell type is or if there are large changes in expression in less frequent cells, but will completely mask changes exhibited by rare cells. One limiting factor in our ability to study more levels of heterogeneity in leukemia subpopulations is the lack of appropriately powerful and sensitive techniques to assay more complex facets of cellular behavior in such small populations. We have developed several multiplexed, high sensitivity analysis techniques and applied them to study multiple facets of cell behavior and identity in rare cell samples, down to the single cell level. By analyzing expression and genotype in smaller samples, direct from cells, we can analyze heterogeneity in expression and alternative splicing, combined with both cell surface markers and the presence of multiple mutations. These innovative techniques have allowed us to study clonality in AML samples in detail via a robust assay that can identify multiple mutations occurring concurrently in single cells. We have also optimized a protocol to simultaneously assay gene expression of 24 in 100-500 cell samples from flow sorted leukemia cell populations. Finally multiplexed assays for alternative splice variant detection in single cells has enabled us to study whether alternative splice variant expression of several genes occurs concurrently in the same cells, or in different cells of the population. With these techniques in hand we have the unique opportunity to begin studying the biology of these diseases from a previously unrealistic angle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3686. doi:1538-7445.AM2012-3686

Overexpression of carbonic anhydrase and HIF-1α in Wilms tumours

BackgroundOverexpression of carbonic anhydrase (CA IX) is associated with poor survival in several adult-type cancers but its expression is undocumented in Wilms tumour (WT), the most common tumour of the paediatric kidney.MethodsCA9 expression was measured using polymerase chain reaction (PCR) in 13 WTs and matched-paired non-neoplastic kidneys (NKs). CA IX and hypoxia-inducible factor-1 α-subunit (HIF-1α) protein were quantified in 15 matched-paired WTs and NKs using enzyme-linked immunosorbent assays. CA IX and HIF-1α were localised by immunostaining tissue sections of 70 WTs (untreated WTs, n = 22; chemotherapy-treated WTs, n = 40; relapsed/metastatic WTs, n = 8). CA IX-positive untreated WTs (n = 14) were immunostained for vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1) and CD31. Double staining for CA IX and CD31 was performed in WTs (n = 14).ResultsCA9 full length (FL) was significantly up-regulated in WTs compared to NKs (p = 0.009) by real-time PCR. Conventional PCR showed expression of alternative splice variant in all NKs and WTs but FL in WTs only. WTs showed a 2-fold increase in CA IX protein over NKs (p = 0.01). HIF-1α levels were up-regulated in WTs compared to NKs, although the difference was not statistically significant (p = 0.09). CA IX and HIF-1α immunolocalisation were observed in 63% and 93% of WTs, respectively. The median fraction of cells staining positively for CA IX and HIF-1α was 5% and 22%, respectively. There was no significant association between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. VEGF and GLUT1 immunoreactivity was seen in 94% and 100% with the median fraction of 10% and 60% respectively. Co-expression and co-localisation of all four hypoxia markers was seen in 7/14 and 6/14 cases respectively. CA IX was seen in well vascularised areas as well as in the peri-necrotic areas.ConclusionsCarbonic anhydrase 9 (mRNA and protein), and HIF-1α protein are overexpressed in a significant portion of WTs. No significant association was detected between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. Cellular localisation studies in untreated WTs suggest that CA IX and HIF-1α are regulated by hypoxia and non-hypoxia mechanisms.

Hypoxia inducible factor 1α gene (HIF-1α) splice variants: potential prognostic biomarkers in breast cancer

BackgroundHypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known.MethodsUsing real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival.ResultsHIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1αTAG) and HIF-1α736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1αTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1αTAG (P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1αTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1αTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01).ConclusionsOur results show for the first time that mRNA expression of a HIF-1αTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45

Splicing Diversity of the Human OCLN Gene and Its Biological Significance for Hepatitis C Virus Entry

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.

Multiple forms of BRMS1 are differentially expressed in the MCF10 isogenic breast cancer progression model

Clinical studies evaluating the mRNA expression level of the BRMS1 metastasis suppressor in the progression of breast cancer have not been consistent. The purpose of this study was to characterize endogenous BRMS1 mRNA and protein in a model of the progression of breast cancer. BRMS1 protein expression was evaluated in the genetically related MCF10 cell lines representing 'normal' breast epithelial cells (MCF10A), pre-malignant breast disease (MCF10AT), comedo ductal carcinoma in situ (MCF10DCIS.com), and metastatic carcinoma (MCF10CAa.1 and MCF10CAd.1alpha) with two antibodies that recognize distinct epitopes in the BRMS1 protein. Nuclear expression of the characteristic *35 kDa BRMS1 protein was detected in all cell lines. Because BRMS1 was expressed in the metastatic MCF10 variants, the BRMS1 exons were sequenced to scan for possible genetic mutations. BRMS1 was wild-type with the exception of a synonymous T/C transition in exon 7. However, alternatively spliced variants were detected by RT-PCR. Two variants, BRMS1.v2 and BRMS1.v4 were only detected in the MCF10A and AT cell lines, while BRMS1 and BRMS1.v3 were detected in all lines. These results demonstrate that expression of the characteristic *35 kDa BRMS1 protein is not sufficient to prevent metastasis. The differential expression of alternative splice variants suggests caution should be taken when evaluating BRMS1 mRNA in clinical samples.

Conserved expression of alternative splicing variants of peroxisomalacyl-CoA oxidase 1in vertebrates and developmental and nutritional regulation in fish

The acyl-coenzyme A oxidase 1 (ACOX1) catalyzes the first, rate-limiting step in peroxisomal beta-oxidation of medium to very long straight-chain fatty acids. Zebrafish (Danio rerio) acox1 was characterized and compared with homologs from other sequenced genomes, revealing a remarkable conservation of structure in the vertebrate lineage. Strictly conserved regions of the deduced proteins included acyl-CoA oxidase and FAD binding domains, as well as a COOH-terminal peroxisomal targeting signal. Whole mount in situ hybridization showed that zebrafish acox1 transcripts were diffusely distributed in early-stage embryonic cells, then discreetly expressed in the brain and widely present in the liver and intestine at later stages. An evolutionarily conserved alternative splicing of the corresponding acox1 primary transcript was identified in teleosts and tetrapods including mammals, giving rise, after exon skipping, to two splice variants, ACOX1-3I and ACOX1-3II. Real-time quantitative RT-PCR on zebrafish adult tissues indicated high levels of both variants in the liver, anterior intestine, and to a lesser extent, in the brain. However, the ACOX1-3II transcript variant was expressed seven times more in zebrafish brain than the ACOX1-3I variant. These data suggest a tissue-specific modulation of ACOX1 activity by exchanging exon 3 duplicated isoforms containing amino acid sequences that are potentially implicated in fatty acyl chain specificity. In addition, a significant pretranslational up-regulation of zebrafish and rainbow trout (Oncorhynchus mykiss) acox1 expression was observed in the anterior intestine after feeding. Taken together, these data indicate that ACOX1 alternative splicing isoforms play a key conserved role in the vertebrate fatty acid metabolism.