Alternative Splice Variants Modulates Dominant-Negative Function of Helios in T-Cell Leukemia.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhao S
PROVIDER: S-EPMC5040427 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
Publications
Alternative Splice Variants Modulates Dominant-Negative Function of Helios in T-Cell Leukemia.
Zhao Shaorong S Liu Wei W Li Yinghui Y Liu Pengjiang P Li Shufang S Dou Daolei D Wang Yue Y Yang Rongcun R Xiang Rong R Liu Feifei F
PloS one 20160928 9
The molecular defects which lead to multistep incidences of human T-cell leukemia have yet to be identified. The DNA-binding protein Helios (known as IKZF2), a member of the Ikaros family of Krüppel-like zinc-finger proteins, functions pivotally in T-cell differentiation and activation. In this study, we identify three novel short Helios splice variants which are T-cell leukemic specific, and demonstrate their dominant-negative function. We then test the cellular localization of distinct Helios ...[more]
Similar Datasets
Project description:Alternative pre-mRNA splicing yields functionally distinct splice variants in regulating normal cell differentiation as well as cancer development. The putative tumor suppressor gene GT198 (PSMC3IP), encoding a protein also known as TBPIP and Hop2, has been shown to regulate steroid hormone receptor-mediated transcription and to stimulate homologous recombination in DNA repair. Here, we have identified 6 distinct GT198 splice variant transcripts generated by alternative promoter usage or alternative splicing. Various splice variant transcripts preserve a common open reading frame, which encodes the DNA binding domain of GT198. The splice variants act as dominant negatives to counteract wild-type GT198 activity in transcription and to abolish Rad51 foci formation during radiation-induced DNA damage. In fallopian tube cancer, we have identified 44 point mutations in GT198 clustered in 2 mutation hotspot sequences. The mutation hotspots coincide with the regulatory sequences responsible for alternative splicing, strongly supporting that imbalanced alternative splicing is a selected consequence in cancer. In addition, splice variant-associated cytoplasmic expression is found in tumors carrying germline or somatic GT198 mutations. An altered alternative splicing pattern with increased variants is also present in lymphoblastoid cells derived from familial breast cancer patients carrying GT198 germline mutations. Furthermore, GT198 and its variant are reciprocally expressed during mouse stem cell differentiation. The constitutive expression of the GT198 variant but not the wild type induces tumor growth in nude mice. Our results collectively suggest that mutations in the GT198 gene deregulate alternative splicing. Defective alternative splicing promotes antagonizing variants and in turn induces a loss of the wild type in tumorigenesis. The study highlights the role of alternative splicing in tumor suppressor gene inactivation.
| S-EPMC3743156 | biostudies-literature
Project description:Induction of reactive oxygen species (ROS) is a central mechanism in alcohol hepatotoxicity. Krüppel-like factor 6 (KLF6), a transcription factor and a tumor-suppressor gene, is an early-responsive gene to injury; however, the effect of ROS and alcohol on KLF6 induction is unknown. The aim of this study is to investigate the contribution of 2 sources of ROS, cytochrome P450 2E1 (CYP2E1), NAD(P)H quinone oxidoreductase (NQO1), and alcohol on the modulation of KLF6(Full) expression, splicing to KLF6_V1 and KLF6_V2, and the effect on TNF?, a downstream target.Endogenous ROS production in CYP2E1-expressing HepG2 cells induced mRNA and protein expression of KLF6(Full) and its splice variants compared to control cells. Incubation with pro-oxidants such as arachidonic acid (AA), ?-naphtoflavone, and H(2) O(2) further enhanced KLF6(Full) and its splice variants. The AA effects on KLF6(Full) and its splice forms were blocked by vitamin E-which prevents lipid peroxidation-and by diallylsulfide-a CYP2E1 inhibitor. Menadione and paraquat, 2 pro-oxidants metabolized via NQO1, induced KLF6(Full) mRNA in a thiol-dependent manner. Antioxidants and an NQO1 inhibitor suppressed the menadione-dependent increase in KLF6(Full) and its splice variants mRNA. Furthermore, primary hepatocytes and livers from chronic alcohol-fed rats, with elevated lipid peroxidation, H(2) O(2) and CYP2E1 but with low GSH, showed a ~2-fold increase in KLF6(Full) mRNA compared to controls. Inhibition of p38 phosphorylation further up-regulated the CYP2E1 and the AA effects on KLF6(Full) mRNA, whereas inhibition JNK and ERK1/2 phosphorylation decreased both. KLF6_V1 but not KLF6(Full) ablation markedly increased TNF? levels in macrophages; thus, TNF? emerges as a downstream target of KLF6_V1.The novel effect of ROS on modulating KLF6(Full) expression and its splice variants could play a relevant role in liver injury and in TNF? regulation.
| S-EPMC3396787 | biostudies-other
Project description:Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.
| S-EPMC4556188 | biostudies-literature
Project description:Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-?E4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.
| S-EPMC4209801 | biostudies-literature
Project description:The capsaicin receptor (TRPV1) is a non-selective cation channel predominantly expressed in specialized sensory neurons that detect painful stimuli. Although its many functional roles continue to be revealed, it has been confirmed to play a critical role in the perception of peripheral inflammatory hyperalgesia and pain. TRPV1 not only is sensitized and/or activated under a wide range of conditions including inflammation and nerve injury but also undergoes changes in expressed levels in response to these same pathologic conditions. Just as our understanding of the structural requirements of TRPV1 activation has grown, there is evidence that TRPV1 forms heteromeric channel complexes. This review is focused on the structural and functional consequence of TRPV1 splice variants: VR.5'sv, TRPV1b/beta and TRPV1var. Through their co-expression and formation of heteromeric complexes with TRPV1, they have been shown to modulate TRPV1 activation. Moreover, TRPV1 splice variant subunits may also contribute unique properties of activation such as the detection of hypertonic conditions.
| S-EPMC3888216 | biostudies-literature
Project description:CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors.Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL.The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.
| S-EPMC4774009 | biostudies-other
Project description:PurposeUp to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity.MethodsWe identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database.ResultsIn heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019).ConclusionMost SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.
| S-EPMC9262418 | biostudies-literature
Project description:BackgroundTalins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene.ResultsWe show here that, based on their distribution in cDNA libraries, Talin-a and Talin-b are differentially expressed during C. intestinalis development. The I/LWEQ modules of the two proteins also have different affinities for F-actin. Consistent with the hypothesis that Talin-a and Talin-b have different roles in cell adhesion, the distinct I/LWEQ modules of Talin-a and Talin-b possess different subcellular targeting determinants. The I/LWEQ module of Talin-a is targeted to focal adhesions, where it most likely serves as the link between integrin and the actin cytoskeleton. The Talin-b I/LWEQ module is not targeted to focal adhesions, but instead preferentially labels F-actin stress fibers. These different properties of C. intestinalis the Talin-a and Talin-b I/LWEQ modules mimic the differences between mammalian Talin1 and Talin2.ConclusionVertebrates and D. discoideum contain two talin genes that encode proteins with different functions. The urochordate C. intestinalis has a single talin gene but produces two separate talins by alternative splicing that vary in a domain crucial for talin function. This suggests that multicellular organisms require multiple talins as components of adhesion complexes. In C. intestinalis, alternative splicing, rather than gene duplication followed by neo-functionalization, accounts for the presence of multiple talins with different properties. Given that C. intestinalis is an excellent model system for chordate biology, the study of Talin-a and Talin-b will lead to a deeper understanding of cell adhesion in the chordate lineage and how talin functions have been parceled out to multiple proteins during metazoan evolution.
| S-EPMC1702346 | biostudies-literature
Project description:The efficacy of anti-angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti-VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open-reading frame in previously described splice variants of VEGF (VEGFXXX ), leading to a change in the C-terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGFXXX/NF ) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF222/NF (equivalent to VEGF165 ) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF222/NF in mice, which we treated with polyclonal anti-VEGFXXX/NF antibodies. VEGF222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti-VEGFXXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGFXXX/NF levels, resistance to anti-VEGFR therapy and survival. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti-angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti-VEGFR therapy.
| S-EPMC10323879 | biostudies-literature
Project description:The telomerase catalytic subunit (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a "deletion" variant (hTERTalpha) that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTalpha inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity.
| S-EPMC1507985 | biostudies-literature