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Sample records for active ar splice

  1. AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression.

    PubMed

    Li, Y; Hwang, T H; Oseth, L A; Hauge, A; Vessella, R L; Schmechel, S C; Hirsch, B; Beckman, K B; Silverstein, K A; Dehm, S M

    2012-11-08

    Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

  2. Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells.

    PubMed

    Shafi, Ayesha A; Putluri, Vasanta; Arnold, James M; Tsouko, Efrosini; Maity, Suman; Roberts, Justin M; Coarfa, Cristian; Frigo, Daniel E; Putluri, Nagireddy; Sreekumar, Arun; Weigel, Nancy L

    2015-10-13

    Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

  3. AR Alternative Splicing and Prostate Cancer Progression

    DTIC Science & Technology

    2012-07-01

    the PCR target regions . For conventional PCR, genomic DNA was amplified using a Taq Polymerase PCR kit (Qia- gen), according to the manufacturer’s...n repeats and AT-rich sequence in both of these regions (Fig. 4C). It is common for the endpoints of genomic deletions or insertions to map to...rearrangements under- lying TE- related genetic diseases, including cancer (29), and often arise through NAHR. However, sequencing the 22Rv1 AR break fusion

  4. The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

    PubMed

    Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

    2017-01-29

    Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.

  5. Lin28 Induces Resistance to Anti-Androgens Via Promotion of AR Splice Variant Generation

    PubMed Central

    Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P.; Gao, Allen C.

    2017-01-01

    BACKGROUND Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). METHODS Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. RESULTS We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. CONCLUSIONS Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa

  6. Splicing Factor Prp8 Interacts With NES(AR) and Regulates Androgen Receptor in Prostate Cancer Cells.

    PubMed

    Wang, Dan; Nguyen, Minh M; Masoodi, Khalid Z; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A; Dhir, Rajiv; Wang, Zhou

    2015-12-01

    Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NES(AR)) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NES(AR), we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NES(AR) export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NES(AR) function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NES(AR). Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NES(AR) and regulates AR function in prostate cancer cells.

  7. Kava components down-regulate expression of AR and AR splice variants and reduce growth in patient-derived prostate cancer xenografts in mice.

    PubMed

    Li, Xuesen; Liu, Zhongbo; Xu, Xia; Blair, Christopher A; Sun, Zheng; Xie, Jun; Lilly, Michael B; Zi, Xiaolin

    2012-01-01

    Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5'6'-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.

  8. Kava Components Down-Regulate Expression of AR and AR Splice Variants and Reduce Growth in Patient-Derived Prostate Cancer Xenografts in Mice

    PubMed Central

    Li, Xuesen; Liu, Zhongbo; Xu, Xia; Blair, Christopher A.; Sun, Zheng; Xie, Jun; Lilly, Michael B.; Zi, Xiaolin

    2012-01-01

    Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5′6′-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR. PMID:22347450

  9. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  10. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7

    PubMed Central

    Stockley, Jacqueline; Markert, Elke; Zhou, Yan; Robson, Craig N.; Elliott, David J.; Lindberg, Johan; Leung, Hing Y.; Rajan, Prabhakar

    2015-01-01

    Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa. PMID:26310125

  11. An imaging agent to detect androgen receptor and its active splice variants in prostate cancer

    PubMed Central

    Imamura, Yusuke; Tien, Amy H.; Pan, Jinhe; Leung, Jacky K.; Banuelos, Carmen A.; Jian, Kunzhong; Wang, Jun; Mawji, Nasrin R.; Fernandez, Javier Garcia; Lin, Kuo-Shyan; Andersen, Raymond J.; Sadar, Marianne D.

    2016-01-01

    Constitutively active splice variants of androgen receptor (AR-Vs) lacking ligand-binding domain (LBD) are a mechanism of resistance to androgen receptor LBD–targeted (AR LBD–targeted) therapies for metastatic castration-resistant prostate cancer (CRPC). There is a strong unmet clinical need to identify prostate cancer patients with AR-V–positive lesions to determine whether they will benefit from further AR LBD–targeting therapies or should receive taxanes or investigational drugs like EPI-506 or galeterone. Both EPI-506 (NCT02606123) and galeterone (NCT02438007) are in clinical trials and are proposed to have efficacy against lesions that are positive for AR-Vs. AR activation function-1 (AF-1) is common to the N-terminal domains of full-length AR and AR-Vs. Here, we provide proof of concept for developing imaging compounds that directly bind AR AF-1 to detect both AR-Vs and full-length AR. 123I-EPI-002 had specific binding to AR AF-1, which enabled direct visualization of CRPC xenografts that express full-length AR and AR-Vs. Our findings highlight the potential of 123I-EPI-002 as an imaging agent for the detection of full-length AR and AR-Vs in CRPC. PMID:27525313

  12. Armenian Astronomical Society (ArAS) activities

    NASA Astrophysics Data System (ADS)

    Mickaelian, A. M.

    2016-09-01

    A review on the activities and achievements of Armenian Astronomical Society (ArAS) and Armenian astronomy in general during the last years is given. ArAS membership, ArAS electronic newsletters (ArASNews), ArAS webpage, Annual Meetings, Annual Prize for Young Astronomers (Yervant Terzian Prize) and other awards, international relations, presence in international organizations, local and international summer schools, science camps, astronomical Olympiads and other events, matters related to astronomical education, astronomical heritage, amateur astronomy, astronomy outreach and ArAS further projects are described and discussed.

  13. The 20S Proteasome Splicing Activity Discovered by SpliceMet

    PubMed Central

    Textoris-Taube, Kathrin; Janek, Katharina; Keller, Christin; Henklein, Petra; Kloetzel, Peter Michael; Zaikin, Alexey

    2010-01-01

    The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected. PMID:20613855

  14. Cytoplasmic Drosha activity generated by alternative splicing

    PubMed Central

    Dai, Lisheng; Chen, Kevin; Youngren, Brenda; Kulina, Julia; Yang, Acong; Guo, Zhengyu; Li, Jin; Yu, Peng; Gu, Shuo

    2016-01-01

    RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha. PMID:27471035

  15. Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition.

    PubMed

    Královicová, Jana; Vorechovsky, Igor

    2007-01-01

    Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.

  16. Alternative splicing in cancer: implications for biology and therapy.

    PubMed

    Chen, J; Weiss, W A

    2015-01-02

    Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

  17. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    PubMed

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects.

  18. Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

    PubMed Central

    Bourgeois, Cyril F.; Popielarz, Michel; Hildwein, Georges; Stevenin, James

    1999-01-01

    The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity. PMID:10523623

  19. Cadmium restores in vitro splicing activity inhibited by zinc-depletion.

    PubMed

    Lee, Myeong Jin; Ayaki, Hitoshi; Goji, Junko; Kitamura, Keiko; Nishio, Hisahide

    2006-10-01

    Zinc (Zn)-depletion inhibits the second step of RNA splicing, namely exon-ligation. To investigate the effects of cadmium (Cd) and other metal ions on RNA splicing inhibited by Zn-depletion, we measured in vitro splicing activities in the presence of these metals. Zn-depletion in the splicing reaction mixture was achieved by addition of a Zn-chelator, 1,10-phenanthroline. Cd(II) at 1, 5 and 10 microM restored the splicing activity to 2, 24 and 72% of that in the control reaction mixture, while higher concentrations of Cd(II) decreased the splicing activity, and more than 50 microM Cd(II) showed a complete absence of spliced products. Hg(II) also restored the splicing activity, albeit to a lesser extent, since 5 and 10 microM Hg(II) restored the splicing activity to 3 and 4% of the control value. The other metal ions examined in this study, Co(II), Cu(II), Mg(II) and Mn(II), did not show any restoration of the splicing activity. We concluded that Cd(II) could restore the in vitro splicing activity inhibited by Zn-depletion, although higher concentrations of Cd(II) prevented progress of the RNA splicing reaction. These results suggest that Cd(II) has a bifunctional property regarding RNA splicing, and is stimulatory at low concentrations and inhibitory at high concentrations.

  20. TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs.

    PubMed

    Le Guiner, C; Lejeune, F; Galiana, D; Kister, L; Breathnach, R; Stévenin, J; Del Gatto-Konczak, F

    2001-11-02

    TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.

  1. The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

    NASA Astrophysics Data System (ADS)

    Choudhury, Rajarshi; Roy, Sreerupa Ghose; Tsai, Yihsuan S.; Tripathy, Ashutosh; Graves, Lee M.; Wang, Zefeng

    2014-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.

  2. Design of a Split Intein with Exceptional Protein Splicing Activity

    PubMed Central

    2016-01-01

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  3. Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity

    PubMed Central

    Ukat, M.; Schweikert, H. U.; Hiort, O.; Werner, R.; Drop, S. L. S.; Cools, M.; Hughes, I. A.; Audi, L.; Ahmed, S. F.; Demiri, J.; Rodens, P.; Worch, L.; Wehner, G.; Kulle, A. E.; Dunstheimer, D.; Müller-Roßberg, E.; Reinehr, T.; Hadidi, A. T.; Eckstein, A. K.; van der Horst, C.; Seif, C.; Siebert, R.; Ammerpohl, O.; Holterhus, P.-M.

    2016-01-01

    Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. Setting: The study was conducted at a university hospital endocrine research laboratory. Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). Intervention(s): There were no interventions. Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high

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  5. Regulation of hyaluronidase activity by alternative mRNA splicing.

    PubMed

    Lokeshwar, Vinata B; Schroeder, Grethchen L; Carey, Robert I; Soloway, Mark S; Iida, Naoko

    2002-09-13

    Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Increased concentration of HYAL1-type hyaluronidase correlates with tumor progression and is a marker for grade (G) 2 or 3 bladder cancer. Using bladder tissues and cells, prostate cancer cells, and kidney tissues and performing reverse transcription-PCR, cDNA cloning, DNA sequencing, and in vitro translation, we identified splice variants of HYAL1 and HYAL3. HYAL1v1 variant lacks a 30-amino acid (aa) sequence (301-330) present in HYAL1 protein. HYAL1v1, HYAL1v2 (aa 183-435 present in HYAL1 wild type), HYAL1v3 (aa 1-207), HYAL1v4 (aa 260-435), and HYAL1v5 (aa 340-435) are enzymatically inactive and are expressed in normal tissues/cells and G1 bladder tumor tissues. However, HYAL1 wild type is expressed in G2/G3 tumors and in invasive tumor cells. Stable transfection and HYAL1v1-specific antibody confirmed that the HYAL1 sequence from aa 301 to 330 is critical for hyaluronidase activity. All tumor cells and tissues mainly express HYAL3 variants. HYAL3v1 lacks a 30-aa sequence (299-328) present in HYAL3 protein, that is homologous to the 30-aa HYAL1 sequence. HYAL3v1, HYAL3v2 (aa 251-417 present in HYAL3 wild type), and HYAL3v3 (aa 251-417, but lacking aa 299-328), are enzymatically inactive. Although splicing of a single independent exon generates HYAL1v1 and HYAL3v1, internal exon splicing generates the other HYAL1/HYAL3 variants. These results demonstrate that alternative mRNA splicing controls cellular expression of enzymatically active hyaluronidase and may explain the elevated hyaluronidase levels in bladder/prostate cancer.

  6. RNA splicing and splicing regulator changes in prostate cancer pathology.

    PubMed

    Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

    2017-04-05

    Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

  7. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

    PubMed Central

    Fu, X D; Mayeda, A; Maniatis, T; Krainer, A R

    1992-01-01

    The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro. Images PMID:1454802

  8. ELF5-Mediated AR Activation Regulates Prostate Cancer Progression

    PubMed Central

    Li, Kai; Guo, Yongmin; Yang, Xiong; Zhang, Zhihong; Zhang, Changwen; Xu, Yong

    2017-01-01

    The transcription factor E74-like factor 5 (ELF5) is a potent antioncogene that can prevent epithelial-mesenchymal transition (EMT) and metastasis in prostate cancer (PCa). However, little is known how it suppress the tumor growth and if it can interact with androgen receptor (AR). In this study, we find that the ELF5 is frequently expressed in AR activated PCa cells, where it binds to AR acting as a physiological partner and negatively regulates its transcriptional activity. In addition, the interaction between ELF5 and AR is androgen-dependent. Downregulation of ELF5 by shRNA increases the expression of AR-response genes and the progression of PCa. Moreover, ELF5 is a AR-dependent gene that its expression can be induced by androgen and suppressed by antiandrogen treatment. Notably, forced reduction of ELF5 in LNCaP cells facilitates the binding of AR to ARE in ELF5 gene and enabling its transcription, so that low level ELF5 can turn up its own expression by the negative feedback loop. PMID:28287091

  9. ELF5-Mediated AR Activation Regulates Prostate Cancer Progression.

    PubMed

    Li, Kai; Guo, Yongmin; Yang, Xiong; Zhang, Zhihong; Zhang, Changwen; Xu, Yong

    2017-03-13

    The transcription factor E74-like factor 5 (ELF5) is a potent antioncogene that can prevent epithelial-mesenchymal transition (EMT) and metastasis in prostate cancer (PCa). However, little is known how it suppress the tumor growth and if it can interact with androgen receptor (AR). In this study, we find that the ELF5 is frequently expressed in AR activated PCa cells, where it binds to AR acting as a physiological partner and negatively regulates its transcriptional activity. In addition, the interaction between ELF5 and AR is androgen-dependent. Downregulation of ELF5 by shRNA increases the expression of AR-response genes and the progression of PCa. Moreover, ELF5 is a AR-dependent gene that its expression can be induced by androgen and suppressed by antiandrogen treatment. Notably, forced reduction of ELF5 in LNCaP cells facilitates the binding of AR to ARE in ELF5 gene and enabling its transcription, so that low level ELF5 can turn up its own expression by the negative feedback loop.

  10. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

    PubMed Central

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

    2014-01-01

    Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

  11. Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

    PubMed

    Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

    2014-11-01

    Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing.

  12. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-06-15

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci.

  13. Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels

    PubMed Central

    Kuo, Yu-Hung; Abdullaev, Iskandar F.; Hyzinski-García, María C.; Mongin, Alexander A.

    2014-01-01

    Synopsis The proposed Ca2+-activated Cl− channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK293 cells, and compared its properties to Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl− channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2 expressing cells had no detectable Ca2+-activated Cl− currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modeling of the Best1 protein structure. PMID:24341532

  14. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    PubMed

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants.

  15. The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

    PubMed

    Castillo, Ana F; Orlando, Ulises; Helfenberger, Katia E; Poderoso, Cecilia; Podesta, Ernesto J

    2015-06-15

    The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity.

  16. Compensatory signals associated with the activation of human GC 5' splice sites.

    PubMed

    Kralovicova, Jana; Hwang, Gyulin; Asplund, A Charlotta; Churbanov, Alexander; Smith, C I Edvard; Vorechovsky, Igor

    2011-09-01

    GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.

  17. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position.

    PubMed

    Shen, Manli; Mattox, William

    2012-01-01

    SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.

  18. Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

    PubMed

    Meininger, Isabel; Griesbach, Richard A; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-04-12

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

  19. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    PubMed Central

    Meininger, Isabel; Griesbach, Richard A.; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation. PMID:27068814

  20. Homeodomain Protein Otp and Activity-Dependent Splicing Modulate Neuronal Adaptation to Stress

    PubMed Central

    Amir-Zilberstein, Liat; Blechman, Janna; Sztainberg, Yehezkel; Norton, William H.J.; Reuveny, Adriana; Borodovsky, Nataliya; Tahor, Maayan; Bonkowsky, Joshua L.; Bally-Cuif, Laure; Chen, Alon; Levkowitz, Gil

    2015-01-01

    SUMMARY Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal’s adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing. PMID:22284183

  1. Altered motor activity of alternative splice variants of the mammalian kinesin-3 protein KIF1B.

    PubMed

    Matsushita, Masafumi; Yamamoto, Ruri; Mitsui, Keiji; Kanazawa, Hiroshi

    2009-11-01

    Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

  2. Conservation of context-dependent splicing activity in distant Muscleblind homologs

    PubMed Central

    Oddo, Julia C.; Saxena, Tanvi; McConnell, Ona L.; Berglund, J. Andrew; Wang, Eric T.

    2016-01-01

    The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species. PMID:27557707

  3. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    SciTech Connect

    Hu, Yi Ericsson, Ida Doseth, Berit Liabakk, Nina B. Krokan, Hans E. Kavli, Bodil

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  4. Cell cycle-coupled expansion of AR activity promotes cancer progression.

    PubMed

    McNair, C; Urbanucci, A; Comstock, C E S; Augello, M A; Goodwin, J F; Launchbury, R; Zhao, S G; Schiewer, M J; Ertel, A; Karnes, J; Davicioni, E; Wang, L; Wang, Q; Mills, I G; Feng, F Y; Li, W; Carroll, J S; Knudsen, K E

    2017-03-23

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

  5. Early base-pair fluctuations and the activation of mRNA splicing

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    1991-05-01

    By means of multiprocessed Monte Carlo simulations we study the amplification in time structural fluctuations in sequential RNA folding concomitant with transcription. The simulations allow for an exploration of configuration space subject to the realistic time-constraints of RNA synthesis. The treatment focuses on the splicing YC4 intron as a study case. We show how an early disruption in the folding may result in a terminal structure which is active for splicing, bringing together the two cleavage sites at both ends of the intron.

  6. Cooperative Dynamics of AR and ER Activity in Breast Cancer.

    PubMed

    D'Amato, Nicholas C; Gordon, Michael A; Babbs, Beatrice; Spoelstra, Nicole S; Carson Butterfield, Kiel T; Torkko, Kathleen C; Phan, Vernon T; Barton, Valerie N; Rogers, Thomas J; Sartorius, Carol A; Elias, Anthony; Gertz, Jason; Jacobsen, Britta M; Richer, Jennifer K

    2016-11-01

    Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha-positive (ER(+)) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER(+)/AR(+) breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER(+)/AR(+) patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER(+)/AR(+) primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER.

  7. A model for meteoritic and lunar 40Ar/39Ar age spectra: Addressing the conundrum of multi-activation energies

    NASA Astrophysics Data System (ADS)

    Boehnke, P.; Harrison, T. Mark; Heizler, M. T.; Warren, P. H.

    2016-11-01

    Results of whole-rock 40Ar/39Ar step-heating analyses of extra-terrestrial materials have been used to constrain the timing of impacts in the inner solar system, solidification of the lunar magma ocean, and development of planetary magnetic fields. Despite the importance of understanding these events, the samples we have in hand are non-ideal due to mixed provenance, isotopic disturbances from potentially multiple heating episodes, and laboratory artifacts such as nuclear recoil. Although models to quantitatively assess multi-domain, diffusive 40Ar* loss have long been applied to terrestrial samples, their use on extra-terrestrial materials has been limited. Here we introduce a multi-activation energy, multi-diffusion domain model and apply it to 40Ar/39Ar temperature-cycling, step-heating data for meteoritic and lunar samples. We show that age spectra of extra-terrestrial materials, the Jilin chondrite (K-4) and Apollo 16 lunar breccia (67514 , 43), yielding seemingly non-ideal behavior commonly interpreted as either laboratory artifacts or localized shock heating of pyroxene, are meaningful and can be understood in context of the presence of multi-diffusion domains containing multiple activation energies. Internally consistent results from both the meteoritic and lunar samples reveal high-temperature/short duration thermal episodes we interpret as due to moderate shock heating.

  8. 76 FR 43336 - Agency Information Collection Activities: Form AR-11, Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-20

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form AR-11... Collection under Review: Form AR- 11, Alien's Change of Address Card; OMB Control No. 1615-0007. The... applicable component of the Department of Homeland Security sponsoring this collection: Form AR-11....

  9. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    PubMed

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

  10. A novel prostate cancer therapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variants

    PubMed Central

    Sun, Feng; Indran, Inthrani R.; Zhang, Zhi Wei; Tan, M.H.Eileen; Li, Yu; Lim, Z.L.Ryan; Hua, Rui; Yang, Chong; Soon, Fen-Fen; Li, Jun; Xu, H.Eric; Cheung, Edwin; Yong, Eu-Leong

    2015-01-01

    Persistent androgen receptor (AR) signaling is the key driving force behind progression and development of castration-resistant prostate cancer (CRPC). In many patients, AR COOH-terminal truncated splice variants (ARvs) play a critical role in contributing to the resistance against androgen depletion therapy. Unfortunately, clinically used antiandrogens like bicalutamide (BIC) and enzalutamide (MDV), which target the ligand binding domain, have failed to suppress these AR variants. Here, we report for the first time that a natural prenylflavonoid, icaritin (ICT), can co-target both persistent AR and ARvs. ICT was found to inhibit transcription of key AR-regulated genes, such as KLK3 [prostate-specific antigen (PSA)] and ARvs-regulated genes, such as UBE2C and induce apoptosis in AR-positive prostate cancer (PC) cells. Mechanistically, ICT promoted the degradation of both AR and ARvs by binding to arylhydrocarbon-receptor (AhR) to mediate ubiquitin-proteasomal degradation. Therefore, ICT impaired AR transactivation in PC cells. Knockdown of AhR gene restored AR stability and partially prevented ICT-induced growth suppression. In clinically relevant murine models orthotopically implanted with androgen-sensitive and CRPC cells, ICT was able to target AR and ARvs, to inhibit AR signaling and tumor growth with no apparent toxicity. Our results provide a mechanistic framework for the development of ICT, as a novel lead compound for AR-positive PC therapeutics, especially for those bearing AR splice variants. PMID:25908644

  11. A Novel Androgen Receptor Splice Variant Is Upregulated during Prostate Cancer Progression and Promotes Androgen-depletion-resistant Growth

    PubMed Central

    Guo, Zhiyong; Yang, Xi; Sun, Feng; Jiang, Richeng; Linn, Douglas E.; Chen, Hege; Chen, Hegang; Kong, Xiangtian; Melamed, Jonathan; Tepper, Clifford G.; Kung, Hsing-Jien; Brodie, Angela M. H.; Edwards, Joanne; Qiu, Yun

    2009-01-01

    The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA. PMID:19244107

  12. Alternative Splicing of hTERT Pre-mRNA: A Potential Strategy for the Regulation of Telomerase Activity

    PubMed Central

    Liu, Xuewen; Wang, Yuchuan; Chang, Guangming; Wang, Feng; Wang, Fei; Geng, Xin

    2017-01-01

    The activation of telomerase is one of the key events in the malignant transition of cells, and the expression of human telomerase reverse transcriptase (hTERT) is indispensable in the process of activating telomerase. The pre-mRNA alternative splicing of hTERT at the post-transcriptional level is one of the mechanisms for the regulation of telomerase activity. Shifts in splicing patterns occur in the development, tumorigenesis, and response to diverse stimuli in a tissue-specific and cell type–specific manner. Despite the regulation of telomerase activity, the alternative splicing of hTERT pre-mRNA may play a role in other cellular functions. Modulating the mode of hTERT pre-mRNA splicing is providing a new precept of therapy for cancer and aging-related diseases. This review focuses on the patterns of hTERT pre-mRNA alternative splicing and their biological functions, describes the potential association between the alternative splicing of hTERT pre-mRNA and telomerase activity, and discusses the possible significance of the alternative splicing of the hTERT pre-mRNA in the diagnosis, therapy, and prognosis of cancer and aging-related diseases. PMID:28272339

  13. Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

    PubMed Central

    Kang, Yoon-Suk; Brame, Keenan; Jetter, Jonathan; Bothner, Brian B.; Wang, Gejiao

    2016-01-01

    ABSTRACT ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate

  14. Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

    PubMed

    Ferraldeschi, Roberta; Welti, Jonathan; Powers, Marissa V; Yuan, Wei; Smyth, Tomoko; Seed, George; Riisnaes, Ruth; Hedayat, Somaieh; Wang, Hannah; Crespo, Mateus; Nava Rodrigues, Daniel; Figueiredo, Ines; Miranda, Susana; Carreira, Suzanne; Lyons, John F; Sharp, Swee; Plymate, Stephen R; Attard, Gerhardt; Wallis, Nicola; Workman, Paul; de Bono, Johann S

    2016-05-01

    Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR.

  15. Cell-cycle coupled expansion of AR activity promotes cancer progression

    PubMed Central

    McNair, Christopher; Urbanucci, Alfonso; Comstock, Clay E.S.; Augello, Michael A.; Goodwin, Jonathan F.; Launchbury, Rosalind; Zhao, Shuang; Schiewer, Mathew J.; Ertel, Adam; Karnes, Jeffrey; Davicioni, Elai; Wang, Liguo; Wang, Qianben; Mills, Ian G.; Feng, Felix Y.; Li, Wei; Carroll, Jason S.; Knudsen, Karen E.

    2016-01-01

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle (“Cell Cycle Common”), versus those that were specifically enriched in a subset of cell cycle phases (“Phase Restricted”). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide-desaturase 1 (DEGS1) was identified as an AR regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention, and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention. PMID:27669432

  16. Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation

    SciTech Connect

    Gesnel, Marie-Claude; Theoleyre, Sandrine; Del Gatto-Konczak, Fabienne; Breathnach, Richard . E-mail: breathna@nantes.inserm.fr

    2007-07-13

    In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

  17. Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas

    PubMed Central

    Skubal, Magdalena; Gielen, Gerrit H.; Waha, Anke; Gessi, Marco; Kaczmarczyk, Lech; Seifert, Gerald; Freihoff, Dorothee; Freihoff, Johannes; Pietsch, Torsten; Simon, Matthias; Theis, Martin; Steinhäuser, Christian; Waha, Andreas

    2016-01-01

    Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3′UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas–most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties. PMID:27256982

  18. Alteration of natural (37)Ar activity concentration in the subsurface by gas transport and water infiltration.

    PubMed

    Guillon, Sophie; Sun, Yunwei; Purtschert, Roland; Raghoo, Lauren; Pili, Eric; Carrigan, Charles R

    2016-05-01

    High (37)Ar activity concentration in soil gas is proposed as a key evidence for the detection of underground nuclear explosion by the Comprehensive Nuclear Test-Ban Treaty. However, such a detection is challenged by the natural background of (37)Ar in the subsurface, mainly due to Ca activation by cosmic rays. A better understanding and improved capability to predict (37)Ar activity concentration in the subsurface and its spatial and temporal variability is thus required. A numerical model integrating (37)Ar production and transport in the subsurface is developed, including variable soil water content and water infiltration at the surface. A parameterized equation for (37)Ar production in the first 15 m below the surface is studied, taking into account the major production reactions and the moderation effect of soil water content. Using sensitivity analysis and uncertainty quantification, a realistic and comprehensive probability distribution of natural (37)Ar activity concentrations in soil gas is proposed, including the effects of water infiltration. Site location and soil composition are identified as the parameters allowing for a most effective reduction of the possible range of (37)Ar activity concentrations. The influence of soil water content on (37)Ar production is shown to be negligible to first order, while (37)Ar activity concentration in soil gas and its temporal variability appear to be strongly influenced by transient water infiltration events. These results will be used as a basis for practical CTBTO concepts of operation during an OSI.

  19. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

    PubMed Central

    Ponissery Saidu, Samsudeen; Stephan, Aaron B.; Talaga, Anna K.

    2013-01-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties. PMID:23669718

  20. NF-kappaB2/p52:c-Myc:hnRNPA1 pathway regulates expression of androgen receptor splice variants and enzalutamide sensitivity in prostate cancer

    PubMed Central

    Nadiminty, Nagalakshmi; Tummala, Ramakumar; Liu, Chengfei; Lou, Wei; Evans, Christopher P.; Gao, Allen C.

    2015-01-01

    Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signaling. Alternative splicing of the AR to generate constitutively active, ligand-independent variants is one of the principal mechanisms that promote the development of resistance to next-generation anti-androgens such as enzalutamide. Here, we demonstrate that the splicing factor heterogeneous nuclear RNA-binding protein A1 (hnRNPA1) plays a pivotal role in the generation of AR splice variants such as AR-V7. HnRNPA1 is overexpressed in prostate tumors compared to benign prostates and its expression is regulated by NF-kappaB2/p52 and c-Myc. CRPC cells resistant to enzalutamide exhibit higher levels of NF-kappaB2/p52, c-Myc, hnRNPA1, and AR-V7. Levels of hnRNPA1 and of AR-V7 are positively correlated with each other in PCa. The regulatory circuit involving NF-kappaB2/p52, c-Myc and hnRNPA1 plays a central role in the generation of AR splice variants. Downregulation of hnRNPA1 and consequently of AR-V7 resensitizes enzalutamide-resistant cells to enzalutamide, indicating that enhanced expression of hnRNPA1 may confer resistance to AR-targeted therapies by promoting the generation of splice variants. These findings may provide a rationale for co-targeting these pathways to achieve better efficacy through AR blockade. PMID:26056150

  1. Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

    PubMed Central

    Díaz-Hernández, Juan Ignacio; Sebastián-Serrano, Álvaro; Gómez-Villafuertes, Rosa

    2015-01-01

    P2X receptors are ligand-gated ion channels sensitive to extracellular nucleotides formed by the assembling of three equal or different P2X subunits. In this work we report, for the first time, the accumulation of the P2X6 subunit inside the nucleus of hippocampal neurons in an age-dependent way. This location is favored by its anchorage to endoplasmic reticulum through its N-terminal domain. The extracellular domain of P2X6 subunit is the key to reach the nucleus, where it presents a speckled distribution pattern and is retained by interaction with the nuclear envelope protein spectrin α2. The in vivo results showed that, once inside the nucleus, P2X6 subunit interacts with the splicing factor 3A1, which ultimately results in a reduction of the mRNA splicing activity. Our data provide new insights into post-transcriptional regulation of mRNA splicing, describing a novel mechanism that could explain why this process is sensitive to changes that occur with age. PMID:25874565

  2. A single cell level measurement of StAR expression and activity in adrenal cells.

    PubMed

    Lee, Jinwoo; Yamazaki, Takeshi; Dong, Hui; Jefcoate, Colin

    2017-02-05

    The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kB mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kB StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress.

  3. Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

    PubMed

    Xu, Yilin; Gao, Xin D; Lee, Jae-Hyung; Huang, Huilin; Tan, Haiyan; Ahn, Jaegyoon; Reinke, Lauren M; Peter, Marcus E; Feng, Yue; Gius, David; Siziopikou, Kalliopi P; Peng, Junmin; Xiao, Xinshu; Cheng, Chonghui

    2014-06-01

    Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

  4. [vWF improves secretion and activity of intein spliced BDD-FVIII].

    PubMed

    Zhu, Fu-Xiang; Yang, Shu-De; Liu, Ze-Long; Miao, Jing; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-05-01

    As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

  5. Bovine Herpesvirus 4 Modulates Its β-1,6-N-Acetylglucosaminyltransferase Activity through Alternative Splicing

    PubMed Central

    Lété, Céline; Markine-Goriaynoff, Nicolas; Machiels, Bénédicte; Pang, Poh-Choo; Xiao, Xue; Canis, Kevin; Suzuki, Masami; Fukuda, Minoru; Dell, Anne; Haslam, Stuart M.; Vanderplasschen, Alain

    2015-01-01

    ABSTRACT Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein β-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus. IMPORTANCE Viruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such

  6. Fluctuations and resulting competing pathways in RNA folding: The activation of splicing

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    1991-01-01

    We implement a parallel processing Monte Carlo simulation to explore RNA configuration space that takes into account fluctuations in base-pairing patterns. The choice of folding pathways is biased by the refolding events that occur as the chain is being assembled. We prove that fluctuations in the initial stages of folding might lead to either active or inactive emerging structures. As an illustration, competing pathways that are the result of fluctuation propagation are computed for the splicing YC4 intron (a segment of the mitochondrial RNA from fungi), and the emerging structures are proved to be biologically relevant.

  7. Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

    PubMed

    Joshi, Jugal Bharat; Patel, Divya; Morton, Derrick J; Sharma, Pankaj; Zou, Jin; Hewa Bostanthirige, Dhanushka; Gorantla, Yamini; Nagappan, Peri; Komaragiri, Shravan Kumar; Sivils, Jeffrey C; Xie, Huan; Palaniappan, Ravi; Wang, Guangdi; Cox, Marc B; Chaudhary, Jaideep

    2016-11-27

    Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.

  8. Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

    PubMed Central

    Sidarovich, Anzhalika; Will, Cindy L; Anokhina, Maria M; Ceballos, Javier; Sievers, Sonja; Agafonov, Dmitry E; Samatov, Timur; Bao, Penghui; Kastner, Berthold; Urlaub, Henning; Waldmann, Herbert; Lührmann, Reinhard

    2017-01-01

    Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation. DOI: http://dx.doi.org/10.7554/eLife.23533.001 PMID:28300534

  9. Fas-activated serine/threonine kinase (FAST K) synergizes with TIA-1/TIAR proteins to regulate Fas alternative splicing.

    PubMed

    Izquierdo, José M; Valcárcel, Juan

    2007-01-19

    The factors and mechanisms that mediate the effects of intracellular signaling cascades on alternative pre-mRNA splicing are poorly understood. TIA-1 (T-cell intracellular antigen 1) and TIAR (TIA-1-related) proteins regulate alternative pre-mRNA splicing by promoting the use of suboptimal 5' splice sites followed by uridine-rich intronic enhancer sequences. These proteins promote, for example, inclusion of Fas receptor exon 6, which leads to an mRNA encoding a pro-apoptotic form of the receptor at the expense of the form that skips exon 6, which encodes an anti-apoptotic form. Fas-activated serine/threonine kinase (FAST K) is known to interact with and phosphorylate TIA-1. Here we have tested the possibility that FAST K influences alternative pre-mRNA splicing by affecting the activity of TIA-1/TIAR. Depletion of FAST K form Jurkat cells leads to skipping of exon 6 from endogenous Fas transcripts. Conversely, FAST K overexpression enhances exon 6 inclusion of Fas reporters transfected in HeLa cells. Consistent with the possibility that the effects of FAST K are mediated by changes in the function of TIA-1/TIAR, the effects of FAST K overexpression (i) are largely suppressed by depletion of TIA-1 and TIAR and (ii) are significantly compromised by mutation of a TIA-1/TIAR-responsive enhancer present downstream of exon 6 5' splice site. Furthermore, in vitro phosphorylation of TIA-1 by FAST K results in enhanced U1 snRNP recruitment. Interestingly, this enhancement is not due to increased binding of TIA-1 to the pre-mRNA. Taken together, the results connect Fas signaling with the activity of splicing factors that modulate Fas alternative splicing, suggesting the existence of an autoregulatory loop that could serve to amplify Fas responses.

  10. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  11. A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions.

    PubMed

    Jagla, Monika; Fève, Marie; Kessler, Pascal; Lapouge, Gaëlle; Erdmann, Eva; Serra, Sebastian; Bergerat, Jean-Pierre; Céraline, Jocelyn

    2007-09-01

    The androgen receptor (AR) is a ligand-activated transcription factor that displays genomic actions characterized by binding to androgen-response elements in the promoter of target genes as well as nongenomic actions that do not require nuclear translocation and DNA binding. In this study, we report exclusive cytoplasmic actions of a splicing variant of the AR detected in a metastatic prostate cancer. This AR variant, named AR23, results from an aberrant splicing of intron 2, wherein the last 69 nucleotides of the intronic sequence are retained, leading to the insertion of 23 amino acids between the two zinc fingers in the DNA-binding domain. We show that the nuclear entry of AR23 upon dihydrotestosterone (DHT) stimulation is impaired. Alternatively, DHT-activated AR23 forms cytoplasmic and perinuclear aggregates that partially colocalize with the endoplasmic reticulum and are devoid of genomic actions. However, in LNCaP cells, this cytoplasmic DHT-activated AR23 remains partially active as evidenced by the activation of transcription from androgen-responsive promoters, the stimulation of NF-kappaB transcriptional activity and by the decrease of AP-1 transcriptional activity. Our data reveal novel cytoplasmic actions for this splicing AR variant, suggesting a contribution in prostate cancer progression.

  12. Alternative splicing regulates activation-induced cytidine deaminase (AID): implications for suppression of AID mutagenic activity in normal and malignant B cells

    PubMed Central

    Wu, Xiaosheng; Darce, Jaime R.; Chang, Sook Kyung; Nowakowski, Grzegorz S.

    2008-01-01

    The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and, currently, it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells, and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly, our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells. PMID:18684869

  13. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  14. Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation.

    PubMed

    Finley, Jahahreeh

    2014-11-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by an accelerated aging phenotype and an average life span of 13years. Patients typically exhibit extensive pathophysiological vascular alterations, eventually resulting in death from stroke or myocardial infarction. A silent point mutation at position 1824 (C1824T) of the LMNA gene, generating a truncated form of lamin A (progerin), has been shown to be the cause of most cases of HGPS. Interestingly, this mutation induces the use of an internal 5' cryptic splice site within exon 11 of the LMNA pre-mRNA, leading to the generation of progerin via aberrant alternative splicing. The serine-arginine rich splicing factor 1 (SRSF1 or ASF/SF2) has been shown to function as an oncoprotein and is upregulated in many cancers and other age-related disorders. Indeed, SRSF1 inhibition results in a splicing ratio in the LMNA pre-mRNA favoring lamin A production over that of progerin. It is our hypothesis that activation of AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, may lead to a reduction in SRSF1 and thus a decrease in the use of the LMNA 5' cryptic splice site in exon 11 through upregulation of p32, a splicing factor-associated protein and putative mitochondrial chaperone that has been shown to inhibit SRSF1 and enhance mitochondrial DNA (mtDNA) replication and oxidative phosphorylation. AMPK activation by currently available compounds such as metformin, resveratrol, and berberine may thus have wide-ranging implications for disorders associated with increased production and accumulation of progerin.

  15. Regulation of Human p53 Activity and Cell Localization by Alternative Splicing

    PubMed Central

    Ghosh, Anirban; Stewart, Deborah; Matlashewski, Greg

    2004-01-01

    The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization. PMID:15340061

  16. Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

    PubMed

    Osipovich, Anna B; White-Grindley, Erica K; Hicks, Geoffrey G; Roshon, Michael J; Shaffer, Christian; Moore, Jason H; Ruley, H Earl

    2004-01-01

    Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

  17. Activation of cryptic 3′ splice sites within introns of cellular genes following gene entrapment

    PubMed Central

    Osipovich, Anna B.; White-Grindley, Erica K.; Hicks, Geoffrey G.; Roshon, Michael J.; Shaffer, Christian; Moore, Jason H.; Ruley, H. Earl

    2004-01-01

    Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3′-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3′ splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3′ splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3′ splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3′ splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3′ splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3′ splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3′ processing and polyadenylation of cellular transcripts. PMID:15155860

  18. Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells

    PubMed Central

    Paronetto, Maria Paola; Messina, Valeria; Barchi, Marco; Geremia, Raffaele; Richard, Stéphane; Sette, Claudio

    2011-01-01

    Sam68 plays an essential role in mouse spermatogenesis and male fertility. Herein, we report an interaction between Sam68 and the phosphorylated forms of the RNA polymerase II (RNAPII) in meiotic spermatocytes. RNase treatment decreased but did not abolish the interaction, consistently with in vitro binding of RNAPII to the Sam68 carboxyl-terminal region. Sam68 retention in the spermatocyte nucleus was dependent on the integrity of cellular RNAs, suggesting that the protein is recruited to transcriptionally active chromatin. Mouse knockout models characterized by stage-specific arrest of spermatogenesis and staining with the phosphorylated form of RNAPII documented that Sam68 expression is confined to the transcriptionally active stages of spermatogenesis. Furthermore, Sam68 associates with splicing regulators in germ cells and we report that alternative splicing of Sgce exon 8 is regulated in a Sam68-dependent manner during spermatogenesis. RNA and chromatin crosslink immunoprecipitation experiments showed that Sam68 binds in vivo to sequences surrounding the intron 7/exon 8 boundary, thereby affecting the recruitment of the phosphorylated RNAPII and of the general splicing factor U2AF65. These results suggest that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells and provide new insights into the regulation of Sam68 expression during spermatogenesis. PMID:21355037

  19. Role of androgen receptor splice variants in prostate cancer metastasis

    PubMed Central

    Xu, Jin; Qiu, Yun

    2017-01-01

    Prostate cancer (PCa) is one of the most lethal cancers in western countries. Androgen receptor (AR) signaling pathway plays a key role in PCa progression. Despite the initial effectiveness of androgen deprivation therapy (ADT) for treatment of patients with advanced PCa, most of them will develop resistance to ADT and progress to metastatic castration resistant prostate cancer (mCRPC). Constitutively transcriptional activated AR splice variants (AR-Vs) have emerged as critical players in the development and progression of mCRPC. Among AR-Vs identified to date, AR-V7 (a.k.a. AR3) is one of the most abundant and frequently found in both PCa cell lines and in human prostate tissues. Most of functional studies have been focused on AR-V7/AR3 and revealed its role in regulation of survival, growth, differentiation and migration in prostate cells. In this review, we will summarize our current understanding of regulation of expression and activity of AR-Vs in mCRPC. PMID:28239558

  20. The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

    PubMed Central

    Malone, Colin D.; Mestdagh, Claire; Akhtar, Junaid; Kreim, Nastasja; Deinhard, Pia; Sachidanandam, Ravi; Treisman, Jessica

    2014-01-01

    The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. PMID:25104425

  1. Plasma parameters and active species kinetics in CF4/O2/Ar gas mixture: effects of CF4/O2 and O2/Ar mixing ratios

    NASA Astrophysics Data System (ADS)

    Lee, Junmyung; Kwon, Kwang-Ho; Efremov, A.

    2016-12-01

    The effects of both CF4/O2 and O2/Ar mixing ratios in three-component CF4/O2/Ar mixture on plasma parameters, densities and fluxes of active species determining the dry etching kinetics were analyzed. The investigation combined plasma diagnostics by Langmuir probes and zero-dimensional plasma modeling. It was found that the substitution of CF4 for O2 at constant fraction of Ar in a feed gas produces the non-monotonic change in F atom density, as it was repeatedly reported for the binary CF4/O2 gas mixtures. At the same time, the substitution of Ar for O2 at constant fraction of CF4 results in the monotonic increase in F atom density toward more oxygenated plasmas. The natures of these phenomena as well as theirs possible impacts on the etching/polymerization kinetics were discussed in details.

  2. Transposon insertions causing constitutive sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing

    SciTech Connect

    Berstein, M.; Cline, T.W. |; Lersch, R.A.; Subrahmanyan, L.

    1995-02-01

    Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle {open_quotes}on{close_quotes} vs. {open_quotes}off{close_quotes} reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. {open_quotes}Male-lethal{close_quotes} (Sxl{sup M}) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, Sxl{sup M} haplo-X animals (chromosomal males) die because of improper dosage compensation, and Sxl{sup m} chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Transcript analysis of double-mutant male-viable Sxl{sup M} derivatives in which the Sxl{sup M} insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these Sxl{sup M} alleles is a consequence of an alteration of the structure of the pre-mRNA that allow some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of Sxl{sup M} alleles appears to depend on the mutant alleles` responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce. 47 refs., 10 figs., 4 tabs.

  3. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    PubMed

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-05

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

  4. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production

    SciTech Connect

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-07

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions. - Highlights: • AR-42 is anti-proliferative against primary/established colon cancer cells. • AR-42 induces significant apoptotic death in primary/established colon cancer cells. • Ceramide production mediates AR-42-induced cytotoxicity in colon cancer cells. • AR-42 oral administration potently inhibits SW-620 xenograft growth in SCID mice.

  5. An alternatively spliced IL-15 isoform modulates abrasion-induced keratinocyte activation.

    PubMed

    Lee, Tsung-Lin; Chang, Mei-Ling; Lin, Yu-Jei; Tsai, Ming-Hsun; Chang, Yi-Hsuan; Chuang, Che-Ming; Chien, Yun; Sosinowski, Tomasz; Wang, Chih-Hsiu; Chen, Yi-Yuan; Lee, Chien-Kuo; Chen, Jau-Shiuh; Wang, Li-Fang; Kung, John T; Ku, Chia-Chi

    2015-05-01

    In a routine phenotype-driven screen, we identified a point mutation in exon 7 of the IL-15 gene in Pedigree 191 (deficient memory (DM)) of N-ethyl-N-nitrosourea mutagenized mice. The DM epidermis expressed an alternatively spliced IL-15 mRNA isoform, IL-15ΔE7, and a wild-type (WT) IL-15 isoform at comparable levels. Mechanical stimulation of DM skin or DM skin graft transplanted onto the WT host resulted in reduced keratinocyte activation and inhibition of neutrophil infiltration into the dermis, demonstrating that DM keratinocytes produced less inflammatory response to external stimulation. Ectopic expression of IL-15ΔE7 in WT skin prevented abrasion-induced epidermal thickening, blocked the accumulation of nuclear antigen Ki67(+) cells in the basal and the suprabasal cell layers, increased loricrin expression, and also increased keratinocyte CXCL1 and G-CSF production. IL-15ΔE7 also profoundly blocked neutrophil infiltration in SDS- or immiquimod (IMQ)-treated WT skin. Recombinant IL-15ΔE7 failed to activate STAT-5 and its downstream target bcl-2 expression. Our study points to IL-15ΔE7 as a potential therapeutic agent for treating neutrophilia-associated inflammatory skin disorders.

  6. Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity

    SciTech Connect

    Wada, Akihiro; Wong, Pooi-Fong; Hojo, Hironobu; Hasegawa, Makoto; Ichinose, Akitoyo; Llanes, Rafael; Kubo, Yoshinao; Senba, Masachika; Ichinose, Yoshio

    2013-05-03

    Highlights: • Alarin inhibits the growth of E. coli but not S. aureus. • Alarin’s potency is comparable to LL-37 in inhibiting the growth of E. coli. • Alarin can cause bacterial membrane blebbing. • Alalin does not induce hemolysis on erythrocytes. -- Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.

  7. Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis

    PubMed Central

    Li, Yi; Zheng, Jia-Yi; Liu, Jian-Qun; Yang, Jie; Liu, Yang; Wang, Chen; Ma, Xiao-Nan; Liu, Bao-Lin; Xin, Gui-Zhong; Liu, Li-Fang

    2016-01-01

    Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-β (TGF-β1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-β1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-β1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1β further increased TGF-β1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-β1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis. PMID:28003810

  8. Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron.

    PubMed

    Echeverria, Gloria V; Cooper, Thomas A

    2014-02-01

    Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5' splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5' splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.

  9. Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

    PubMed Central

    Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

    2016-01-01

    Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest. DOI: http://dx.doi.org/10.7554/eLife.19743.001 PMID:27882870

  10. The 35S U5 snRNP Is Generated from the Activated Spliceosome during In vitro Splicing

    PubMed Central

    2015-01-01

    Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing. PMID:26020933

  11. Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

    PubMed Central

    Topilina, Natalya I.; Novikova, Olga; Stanger, Matthew; Banavali, Nilesh K.; Belfort, Marlene

    2015-01-01

    Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control. PMID:26101259

  12. The dynamic of the splicing of bZIP60 and the proteins encoded by the spliced and unspliced mRNAs reveals some unique features during the activation of UPR in Arabidopsis thaliana.

    PubMed

    Parra-Rojas, Juan; Moreno, Adrian A; Mitina, Irina; Orellana, Ariel

    2015-01-01

    The unfolded protein response (UPR) is a signaling pathway that is activated when the workload of the endoplasmic reticulum (ER) is surpassed. IRE1 is a sensor involved in triggering the UPR and plays a key role in the unconventional splicing of an mRNA leading to the formation of a transcription factor that up-regulates the transcription of genes that play a role in restoring the homeostasis in the ER. In plants, bZIP60 is the substrate for IRE1; however, questions such as what is the dynamics of the splicing of bZIP60 and the fate of the proteins encoded by the spliced and unspliced forms of the mRNA, remain unanswered. In the present work, we analyzed the processing of bZIP60 by determining the levels of the spliced form mRNA in plants exposed to different conditions that trigger UPR. The results show that induction of ER stress increases the content of the spliced form of bZIP60 (bZIP60s) reaching a maximum, that depending on the stimuli, varied between 30 min or 2 hrs. In most cases, this was followed by a decrease in the content. In contrast to other eukaryotes, the splicing never occurred to full extent. The content of bZIP60s changed among different organs upon induction of the UPR suggesting that splicing is regulated differentially throughout the plant. In addition, we analyzed the distribution of a GFP-tagged version of bZIP60 when UPR was activated. A good correlation between splicing of bZIP60 and localization of the protein in the nucleus was observed. No fluorescence was observed under basal conditions, but interestingly, the fluorescence was recovered and found to co-localize with an ER marker upon treatment with an inhibitor of the proteasome. Our results indicate that the dynamics of bZIP60, both the mRNA and the protein, are highly dynamic processes which are tissue-specific and stimulus-dependent.

  13. Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

    DTIC Science & Technology

    2013-07-01

    Stanchina lab, new baselines for tumor growth were derived in 8-10 weeks old athymic female mice, which were subcutaneously inoculated with 10X106 MDA-MB...the left ventricle) or orthotopically into axillary mammary fat pads of 6-week old female athymic nude mice are underway in the de Stanchina lab...splicing and the addition of a 5’ cap and a 3’ polyadenylation sequence [15]. The choice and usage of splice sites in the pre- mRNA can be

  14. Dual-Targeting of AR and Akt Pathways by Berberine in Castration-Resistant Prostate Cancer

    DTIC Science & Technology

    2015-08-01

    prostate cancer driven by a constitutively active AR splice variant. While berberine treatment significantly inhibited the growth of tumor xenografts...or AR-V- promoted CRPC growth . BBR sensitizes ARv567es-expressing tumor xenografts to enzalutamide treatment. In the previous annual report...weeks before the animals were sacrificed. As shown in Fig. 1, the antiandrogen enzalutamide did not significantly change the growth of ARv567es

  15. Dual-Targeting of AR and Akt Pathways by Berberine in Castration-Resistant Prostate Cancer

    DTIC Science & Technology

    2015-08-01

    weeks before the animals were sacrificed. As shown in Fig. 1, the antiandrogen enzalutamide did not significantly change the growth of ARv567es...prostate cancer driven by a constitutively active AR splice variant. While berberine treatment significantly inhibited the growth of tumor xenografts...or AR-V- promoted CRPC growth . BBR sensitizes ARv567es-expressing tumor xenografts to enzalutamide treatment. In the previous annual report

  16. 40Ar/39Ar dating of tuff vents in the Campi Flegrei caldera (southern Italy): Toward a new chronostratigraphic reconstruction of the Holocene volcanic activity

    USGS Publications Warehouse

    Fedele, L.; Insinga, D.D.; Calvert, A.T.; Morra, V.; Perrotta, A.; Scarpati, C.

    2011-01-01

    The Campi Flegrei hosts numerous monogenetic vents inferred to be younger than the 15 ka Neapolitan Yellow Tuff. Sanidine crystals from the three young Campi Flegrei vents of Fondi di Baia, Bacoli and Nisida were dated using 40Ar/39Ar geochronology. These vents, together with several other young edifices, occur roughly along the inner border of the Campi Flegrei caldera, suggesting that the volcanic conduits are controlled by caldera-bounding faults. Plateau ages of ∼9.6 ka (Fondi di Baia), ∼8.6 ka (Bacoli) and ∼3.9 ka (Nisida) indicate eruptive activity during intervals previously interpreted as quiescent. A critical revision, involving calendar age correction of literature 14C data and available 40Ar/39Ar age data, is presented. A new reference chronostratigraphic framework for Holocene Phlegrean activity, which significantly differs from the previously adopted ones, is proposed. This has important implications for understanding the Campi Flegrei eruptive history and, ultimately, for the evaluation of related volcanic risk and hazard, for which the inferred history of its recent activity is generally taken into account.

  17. A splice variant in the ACSL5 gene relates migraine with fatty acid activation in mitochondria

    PubMed Central

    Matesanz, Fuencisla; Fedetz, María; Barrionuevo, Cristina; Karaky, Mohamad; Catalá-Rabasa, Antonio; Potenciano, Victor; Bello-Morales, Raquel; López-Guerrero, Jose-Antonio; Alcina, Antonio

    2016-01-01

    Genome-wide association studies (GWAS) in migraine are providing the molecular basis of this heterogeneous disease, but the understanding of its aetiology is still incomplete. Although some biomarkers have currently been accepted for migraine, large amount of studies for identifying new ones is needed. The migraine-associated variant rs12355831:A>G (P=2 × 10−6), described in a GWAS of the International Headache Genetic Consortium, is localized in a non-coding sequence with unknown function. We sought to identify the causal variant and the genetic mechanism involved in the migraine risk. To this end, we integrated data of RNA sequences from the Genetic European Variation in Health and Disease (GEUVADIS) and genotypes from 1000 GENOMES of 344 lymphoblastoid cell lines (LCLs), to determine the expression quantitative trait loci (eQTLs) in the region. We found that the migraine-associated variant belongs to a linkage disequilibrium block associated with the expression of an acyl-coenzyme A synthetase 5 (ACSL5) transcript lacking exon 20 (ACSL5-Δ20). We showed by exon-skipping assay a direct causality of rs2256368-G in the exon 20 skipping of approximately 20 to 40% of ACSL5 RNA molecules. In conclusion, we identified the functional variant (rs2256368:A>G) affecting ACSL5 exon 20 skipping, as a causal factor linked to the migraine-associated rs12355831:A>G, suggesting that the activation of long-chain fatty acids by the spliced ACSL5-Δ20 molecules, a mitochondrial located enzyme, is involved in migraine pathology. PMID:27189022

  18. Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRβ2) splice variant in breast cancer

    PubMed Central

    Heckler, Mary M.; Zeleke, Tizita Zewde; Divekar, Shailaja D.; Fernandez, Aileen I.; Tiek, Deanna M.; Woodrick, Jordan; Farzanegan, Alexander; Roy, Rabindra; Üren, Aykut; Mueller, Susette C.; Riggins, Rebecca B.

    2016-01-01

    Breast cancer remains a leading cause of cancer-related death in women, and triple negative breast cancer (TNBC) lacks clinically actionable therapeutic targets. Death in mitosis is a tumor suppressive mechanism that occurs in cancer cells experiencing a defective M phase. The orphan estrogen-related receptor beta (ERRβ) is a key reprogramming factor in murine embryonic and induced pluripotent stem cells. In primates, ERRβ is alternatively spliced to produce several receptor isoforms. In cellular models of glioblastoma, short form (ERRβsf) and beta2 (ERRβ2) splice variants differentially regulate cell cycle progression in response to the synthetic agonist DY131, with ERRβ2 driving arrest in G2/M. The goals of the present study are to determine the cellular function(s) of ligand-activated ERRβ splice variants in breast cancer and evaluate the potential of DY131 to serve as an antimitotic agent, particularly in TNBC. DY131 inhibits growth in a diverse panel of breast cancer cell lines, causing cell death that involves the p38 stress kinase pathway and a bimodal cell cycle arrest. ERRβ2 facilitates the block in G2/M, and DY131 delays progression from prophase to anaphase. Finally, ERRβ2 localizes to centrosomes and DY131 causes mitotic spindle defects. Targeting ERRβ2 may therefore be a promising therapeutic strategy in breast cancer. PMID:27363015

  19. RBM4a-regulated splicing cascade modulates the differentiation and metabolic activities of brown adipocytes

    PubMed Central

    Lin, Jung-Chun; Lu, Yi-Han; Liu, Yun-Ru; Lin, Ying-Ju

    2016-01-01

    RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a−/− brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis. PMID:26857472

  20. Alternative splicing of human peroxisome proliferator-activated receptor delta (PPARdelta):effects on translation efficiency and trans-activation ability

    PubMed Central

    Lundell, Kerstin; Thulin, Petra; Hamsten, Anders; Ehrenborg, Ewa

    2007-01-01

    Background Peroxisome proliferator-activated receptor delta (PPARδ) is a member of the nuclear receptor superfamily. Numerous studies have aimed at unravelling the physiological role of PPARδ as a transcriptional regulator whereas the regulation of PPARδ gene expression has been less studied. Results The principal transcription start site in the human PPARδ gene identified here is positioned upstream of exon 1, although four alternative 5'-ends related to downstream exons were identified. The demonstration of multiple 5'-UTR splice variants of PPARδ mRNA, with an impact on translation efficiency, suggests a translational regulation of human PPARδ expression. Five untranslated exons identified in this study contribute to the variability among the 5'-UTRs of human PPARδ mRNAs. Moreover, in vitro studies of a 3'-splice transcript encoding a truncated variant of PPARδ (designated PPARδ2) show that this isoform constitutes a potential dominant negative form of the receptor. Conclusion We propose that alternative splicing of human PPARδ constitutes an intrinsic role for the regulation of PPARδ expression and thus activity, and highlight the significance of alternative splicing of this nuclear receptor in physiology and disease. PMID:17705821

  1. High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

    PubMed

    Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

    2016-03-29

    Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

  2. Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels.

    PubMed

    Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

    2014-01-01

    Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

  3. Synthesis and Splice-Redirecting Activity of Branched, Arginine-Rich Peptide Dendrimer Conjugates of Peptide Nucleic Acid Oligonucleotides

    PubMed Central

    2010-01-01

    Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)4−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence. PMID:20879728

  4. Autonomic control network active in Aplysia during locomotion includes neurons that express splice variants of R15-neuropeptides.

    PubMed

    Romanova, Elena V; McKay, Natasha; Weiss, Klaudiusz R; Sweedler, Jonathan V; Koester, John

    2007-01-01

    Splice-variant products of the R15 neuropeptide gene are differentially expressed within the CNS of Aplysia. The goal of this study was to test whether the neurons in the abdominal ganglion that express the peptides encoded by this gene are part of a common circuit. Expression of R15 peptides had been demonstrated previously in neuron R15. Using a combination of immunocytochemical and analytical methods, this study demonstrated that R15 peptides are also expressed in heart exciter neuron RB(HE), the two L9(G) gill motoneurons, and L40--a newly identified interneuron. Mass spectrometric profiling of individual neurons that exhibit R15 peptide-like immunoreactivity confirmed the mutually exclusive expression of two splice-variant forms of R15 peptides in different neurons. The L9(G) cells were found to co-express pedal peptide in addition to the R15 peptides. The R15 peptide-expressing neurons examined here were shown to be part of an autonomic control circuit that is active during fictive locomotion. Activity in this circuit contributes to implementing a central command that may help to coordinate autonomic activity with escape locomotion. Chronic extracellular nerve recording was used to determine the activity patterns of a subset of neurons of this circuit in vivo. These results demonstrate the potential utility of using shared patterns of neuropeptide expression as a guide for neural circuit identification.

  5. Truncated Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α Splice Variant Is Severely Altered in Huntington's Disease

    PubMed Central

    Johri, Ashu; Starkov, Anatoly A.; Chandra, Abhishek; Hennessey, Thomas; Sharma, Abhijeet; Orobello, Sara; Squitieri, Ferdinando; Yang, Lichuan; Beal, M. Flint

    2011-01-01

    Background Reduced peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) gene expression has been observed in striatal cell lines, transgenic mouse models of Huntington's disease (HD), and brain tissue from HD patients. As this protein is a key transcription regulator of the expression of many mitochondrial proteins, these observations strongly support the role of aberrant mitochondrial function in the pathogenesis of HD. The PGC1α protein undergoes posttranslational modifications that affect its transcriptional activity. The N-truncated splice variant of PGC1α (NT-PGC1α) is produced in tissues, but the role of truncated splice variants of PGC1α in HD and in the regulation of mitochondrial gene expression has not been elucidated. Objective To examine the expression and modulation of expression of NT-PGC1α levels in HD. Methods and Results We found that the NT-PGC1α protein, a splice variant of ∼38 kDa, but not full-length PGC1α is severely and consistently altered in human HD brain, human HD myoblasts, mouse HD models, and HD striatal cells. NT-PGC1α levels were significantly upregulated in HD cells and mouse brown fat by physiologically relevant stimuli that are known to upregulate PGC1α gene expression. This resulted in an increase in mitochondrial gene expression and cytochrome c content. Conclusion Our data suggest that NT-PGC1α is an important component of the PGC1α transcriptional network, which plays a significant role in the pathogenesis of HD. Copyright © 2011 S. Karger AG, Basel PMID:21757867

  6. AR-v7 protein expression is regulated by protein kinase and phosphatase.

    PubMed

    Li, Yinan; Xie, Ning; Gleave, Martin E; Rennie, Paul S; Dong, Xuesen

    2015-10-20

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.

  7. Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

    SciTech Connect

    Cizdziel, P.E.; De Mars, M.; Murphy, E.C. Jr.

    1988-04-01

    The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33/sup 0/C or lower than at 37 to 41/sup 0/C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41/sup 0/C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. The authors exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39/sup 0/C. However, after a short (about 30-min) lag following a shift to 33/sup 0/C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA.

  8. AR Alternative Splicing and Prostate Cancer Progression

    DTIC Science & Technology

    2013-07-01

    cells were maintained in RMPI 1640 (Invitrogen) with 10% fetal bovine serum (FBS), 100 units/ml penicillin , and 100g/ml streptomy- cin in a 5% CO2...dihydro- testosterone (DHT; Sigma). Cell growth in RPMI 1640 medium containing 10% charcoal-stripped serum (CSS) 1 nmol/L DHT was monitored by crystal...Res 2002;62:6606–14. 29. Belancio VP, Hedges DJ, Deininger P. Mammalian non-LTR retro- transposons: for better or worse, in sickness and in health

  9. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production.

    PubMed

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-07

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions.

  10. Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3.

    PubMed

    Yun, Hye Jin; Kwon, Jungsun; Seol, Wongi

    2008-10-01

    The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

  11. High-resolution 40Ar/ 39Ar chronostratigraphy of the post-caldera (<20 ka) volcanic activity at Pantelleria, Sicily Strait

    NASA Astrophysics Data System (ADS)

    Scaillet, Stéphane; Rotolo, Silvio G.; La Felice, Sonia; Vita-Scaillet, Grazia

    2011-09-01

    The island of Pantelleria (Sicily Strait), the type locality for pantellerite, has been the locus of major caldera-forming eruptions that culminated, ca. 50 ka ago, in the formation of the Cinque Denti caldera produced by the Green Tuff eruption. The post-caldera silicic activity since that time has been mostly confined inside the caldera and consists of smaller-energy eruptions represented by more than twenty coalescing pantelleritic centers structurally controlled by resurgence and trapdoor faulting of the caldera floor. A high-resolution 40Ar/ 39Ar study was conducted on key units spanning the recent (post-20 ka) intracaldera activity to better characterize the present-day status (and forecast the short-term behavior of) the system based on the temporal evolution of the latest eruptions. The new 40Ar/ 39Ar data capture a long-term (> 15 ka) decline in eruption frequency with a shift in eruptive pace from 3.5 ka -1 to 0.8 ka -1 associated with a prominent paleosol horizon marking the only recognizable volcanic stasis around 12-14 ka. This shift in extraction frequency occurs without major changes in eruptive style, and is paralleled by a subtle trend of decreasing melt differentiation index. We speculate that this decline probably occurred (i) without short-term variations in melt production/differentiation rate in a steady-state compositionally-zoned silicic reservoir progressively tapped deeper through the sequence, and (ii) that it was possibly modulated by outboard eustatic forcing due to the 140 m sea level rise over the past 21 ka. The intracaldera system is experiencing a protracted stasis since 7 ka. Coupled with recent geodetic evidence of deflation and subsidence of the caldera floor, the system appears today to be on a wane with no temporal evidence for a short-term silicic eruption.

  12. Cellular stress and RNA splicing.

    PubMed

    Biamonti, Giuseppe; Caceres, Javier F

    2009-03-01

    In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

  13. Identification of an IRF-1 splicing transcript in APL cells sharing similar transactivation activity of the full length one.

    PubMed

    Lou, Yejiang; Xia, Di; Yu, Mengxia; Tong, Jianhua; Jin, Jie

    2017-03-20

    Interferon regulatory factor-1 (IRF-1) is a member of the interferon regulatory factor family. It acts as a transcriptional activator and plays a critical role in antiviral defense, immune response, cell growth regulation, apoptosis and cell differentiation. Deletions, mutations or aberrant splicing of IRF-1 would result in its functional inactivation, and closely related to the tumorigenesis. In this work, we identified an IRF-1 splicing transcript (IRF-1-s) in all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemia (APL) cell line NB4 cells. It lost the exon 8 and 9 of the full length IRF-1, expressed in numerous cell types and could be induced to expression by ATRA in NB4 cells. It turned out similar biological activity as full length IRF-1 to enhance the transcription of interferon stimulated response element (ISRE)-containing target genes. Identification of IRF-1-s in NB4 cells would be benefit for our further exploring the signaling pathway of ATRA and interferons, as well as the mechanisms of differentiation of APL cells.

  14. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

    PubMed Central

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-01-01

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development. DOI: http://dx.doi.org/10.7554/eLife.09268.001 PMID:26705333

  15. South African Herbal Extracts as Potential Chemopreventive Agents: Screening for Anticancer Splicing Activity.

    PubMed

    Dlamini, Zodwa; Mbita, Zukile; Bates, David O; Bates, David

    2016-01-01

    RT-PCR is an invaluable tool for the detection and characterization of mRNA. Cancer cell lines are treated with crude plant extracts and RNA is extracted and purified with DNase prior to RT-PCR. RT-PCR first-strand cDNA synthesis is done using random primers and can be refrigerated at 4 °C. PCR from the stored cDNA is performed using transcript-specific primers and electrophoresed on a molecular grade agarose gel to separate the splice variants.

  16. Cryo-EM structure of a human spliceosome activated for step 2 of splicing.

    PubMed

    Bertram, Karl; Agafonov, Dmitry E; Liu, Wen-Ti; Dybkov, Olexandr; Will, Cindy L; Hartmuth, Klaus; Urlaub, Henning; Kastner, Berthold; Stark, Holger; Lührmann, Reinhard

    2017-02-16

    Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.

  17. Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein.

    PubMed

    Izquierdo, José M; Valcárcel, Juan

    2007-07-06

    TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.

  18. CUDC-101, a Novel Inhibitor of Full-Length Androgen Receptor (flAR) and Androgen Receptor Variant 7 (AR-V7) Activity: Mechanism of Action and In Vivo Efficacy.

    PubMed

    Sun, Huiying; Mediwala, Sanjay N; Szafran, Adam T; Mancini, Michael A; Marcelli, Marco

    2016-06-01

    Castration-resistant prostate cancer (CRPC) is an androgen receptor (AR)-dependent disease expected to cause the death of more than 27,000 Americans in 2015. There are only a few available treatments for CRPC, making the discovery of new drugs an urgent need. We report that CUDC-101 (an inhibitor od HER2/NEU, EGFR and HDAC) inhibits both the full length AR (flAR) and the AR variant AR-V7. This observation prompted experiments to discover which of the known activities of CUDC-101 is responsible for the inhibition of flAR/AR-V7 signaling. We used pharmacologic and genetic approaches, and found that the effect of CUDC-101 on flAR and AR-V7 was duplicated only by other HDAC inhibitors, or by silencing the HDAC isoforms HDAC5 and HDAC10. We observed that CUDC-101 treatment or AR-V7 silencing by RNAi equally reduced transcription of the AR-V7 target gene, PSA, without affecting viability of 22Rv1 cells. However, when cellular proliferation was used as an end point, CUDC-101 was more effective than AR-V7 silencing, raising the prospect that CUDC-101 has additional targets beside AR-V7. In support of this, we found that CUDC-101 increased the expression of the cyclin-dependent kinase inhibitor p21, and decreased that of the oncogene HER2/NEU. To determine if CUDC-101 reduces growth in a xenograft model of prostate cancer, this drug was given for 14 days to castrated male SCID mice inoculated with 22Rv1 cells. Compared to vehicle, CUDC-101 reduced xenograft growth in a statistically significant way, and without macroscopic side effects. These studies demonstrate that CUDC-101 inhibits wtAR and AR-V7 activity and growth of 22Rv1 cells in vitro and in vivo. These effects result from the ability of CUDC-101 to target not only HDAC signaling, which was associated with decreased flAR and AR-V7 activity, but multiple additional oncogenic pathways. These observations raise the possibility that treatment of CRPC may be achieved by using similarly multi-targeted approaches.

  19. The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice

    PubMed Central

    Jackson, Travis C; Du, Lina; Janesko-Feldman, Keri; Vagni, Vincent A; Dezfulian, Cameron; Poloyac, Samuel M; Jackson, Edwin K; Clark, Robert SB; Kochanek, Patrick M

    2015-01-01

    Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48  to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4  to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2. PMID:25586139

  20. Estimation of (41)Ar activity concentration and release rate from the TRIGA Mark-II research reactor.

    PubMed

    Hoq, M Ajijul; Soner, M A Malek; Rahman, A; Salam, M A; Islam, S M A

    2016-03-01

    The BAEC TRIGA research reactor (BTRR) is the only nuclear reactor in Bangladesh. Bangladesh Atomic Energy Regulatory Authority (BAERA) regulations require that nuclear reactor licensees undertake all reasonable precautions to protect the environment and the health and safety of persons, including identifying, controlling and monitoring the release of nuclear substances to the environment. The primary activation product of interest in terms of airborne release from the reactor is (41)Ar. (41)Ar is a noble gas readily released from the reactor stacks and most has not decayed by the time it moves offsite with normal wind speed. Initially (41)Ar is produced from irradiation of dissolved air in the primary water which eventually transfers into the air in the reactor bay. In this study, the airborne radioisotope (41)Ar generation concentration, ground level concentration and release rate from the BTRR bay region are evaluated theoretically during the normal reactor operation condition by several governing equations. This theoretical calculation eventually minimizes the doubt about radiological safety to determine the radiation level for (41)Ar activity whether it is below the permissible limit or not. Results show that the estimated activity for (41)Ar is well below the maximum permissible concentration limit set by the regulatory body, which is an assurance for the reactor operating personnel and general public. Thus the analysis performed within this paper is so much effective in the sense of ensuring radiological safety for working personnel and the environment.

  1. Resonant proton scattering on 46Ar using the Active-Target Time Projection Chamber

    NASA Astrophysics Data System (ADS)

    Bradt, J.; Ahn, T.; Ayyad Limonge, Y.; Bazin, D.; Beceiro Novo, S.; Carpenter, L.; Kuchera, M. P.; Lynch, W.; Mittig, W.; Rost, S.; Watwood, N.; Barney, J.; Datta, U.; Estee, J.; Gillibert, A.; Manfredi, J.; Morfouace, P.; Perez Loureiro, D.; Pollacco, E.; Sammut, J.; Sweany, S.

    2016-09-01

    A well-known technique for studying the single-particle properties of neutron-rich nuclei is to use resonant proton scattering on a parent nucleus to populate the isobaric analog states of the corresponding neutron-rich nucleus. The locations and amplitudes of these resonances are directly related to the structure of the nucleus of interest by isospin symmetry. We performed an experiment of this type at the National Superconducting Cyclotron Laboratory to commission the recently completed Active-Target Time Projection Chamber (AT-TPC). A 4.6-MeV/u radioactive beam of 46Ar was injected into the AT-TPC. The detector was filled with isobutane gas-which provided the protons for the reaction and served as the tracking medium-and placed inside a 2-T magnetic field. We will present preliminary results from this experiment and discuss the benefits of the active-target method for this type of measurement.

  2. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

  3. Expression of NKp46 Splice Variants in Nasal Lavage Following Respiratory Viral Infection: Domain 1-Negative Isoforms Predominate and Manifest Higher Activity

    PubMed Central

    Shemer-Avni, Yonat; Kundu, Kiran; Shemesh, Avishai; Brusilovsky, Michael; Yossef, Rami; Meshesha, Mesfin; Solomon-Alemayehu, Semaria; Levin, Shai; Gershoni-Yahalom, Orly; Campbell, Kerry S.; Porgador, Angel

    2017-01-01

    The natural killer (NK) cell activating receptor NKp46/NCR1 plays a critical role in elimination of virus-infected and tumor cells. The NCR1 gene can be transcribed into five different splice variants, but the functional importance and physiological distribution of NKp46 isoforms are not yet fully understood. Here, we shed light on differential expression of NKp46 splice variants in viral respiratory tract infections and their functional difference at the cellular level. NKp46 was the most predominantly expressed natural cytotoxicity receptor in the nasal lavage of patients infected with four respiratory viruses: respiratory syncytia virus, adenovirus, human metapneumovirus, or influenza A. Expression of NKp30 was far lower and NKp44 was absent in all patients. Domain 1-negative NKp46 splice variants (i.e., NKp46 isoform d) were the predominantly expressed isoform in nasal lavage following viral infections. Using our unique anti-NKp46 mAb, D2-9A5, which recognizes the D2 extracellular domain, and a commercial anti-NKp46 mAb, 9E2, which recognizes D1 domain, allowed us to identify a small subset of NKp46 D1-negative splice variant-expressing cells within cultured human primary NK cells. This NKp46 D1-negative subset also showed higher degranulation efficiency in term of CD107a surface expression. NK-92 cell lines expressing NKp46 D1-negative and NKp46 D1-positive splice variants also showed functional differences when interacting with targets. A NKp46 D1-negative isoform-expressing NK-92 cell line showed enhanced degranulation activity. To our knowledge, we provide the first evidence showing the physiological distribution and functional importance of human NKp46 splice variants under pathological conditions. PMID:28261217

  4. The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis.

    PubMed

    Koselny, Kristy; Green, Julianne; DiDone, Louis; Halterman, Justin P; Fothergill, Annette W; Wiederhold, Nathan P; Patterson, Thomas F; Cushion, Melanie T; Rappelye, Chad; Wellington, Melanie; Krysan, Damian J

    2016-12-01

    Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity, including activity against yeasts (e.g., Candida albicans, non-albicans Candida spp., Cryptococcus neoformans), molds (e.g., Fusarium, Mucor), and dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with MICs of 2 to 4 μg/ml. AR-12 is also active against azole- and echinocandin-resistant Candida isolates, and subinhibitory AR-12 concentrations increase the susceptibility of fluconazole- and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad-spectrum antifungal activity.

  5. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    SciTech Connect

    Zhuang, Yan; Nguyen, Hong T.; Lasky, Joseph A.; Cao, Subing; Li, Cui; Hu, Jiyao; Guo, Xinyue; Burow, Matthew E.; Shan, Bin

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  6. The importance of non-nuclear AR signaling in prostate cancer progression and therapeutic resistance.

    PubMed

    Zarif, Jelani C; Miranti, Cindy K

    2016-05-01

    The androgen receptor (AR) remains the major oncogenic driver of prostate cancer, as evidenced by the efficacy of androgen deprivation therapy (ADT) in naïve patients, and the continued effectiveness of second generation ADTs in castration resistant disease. However, current ADTs are limited to interfering with AR ligand binding, either through suppression of androgen production or the use of competitive antagonists. Recent studies demonstrate 1) the expression of constitutively active AR splice variants that no longer depend on androgen, and 2) the ability of AR to signal in the cytoplasm independently of its transcriptional activity (non-genomic); thus highlighting the need to consider other ways to target AR. Herein, we review canonical AR signaling, but focus on AR non-genomic signaling, some of its downstream targets and how these effectors contribute to prostate cancer cell behavior. The goals of this review are to 1) re-highlight the continued importance of AR in prostate cancer as the primary driver, 2) discuss the limitations in continuing to use ligand binding as the sole targeting mechanism, 3) discuss the implications of AR non-genomic signaling in cancer progression and therapeutic resistance, and 4) address the need to consider non-genomic AR signaling mechanisms and pathways as a viable targeting strategy in combination with current therapies.

  7. Alternative splicing in the coding region of Ppo-A1 directly influences the polyphenol oxidase activity in common wheat (Triticum aestivum L.).

    PubMed

    Sun, Youwei; He, Zhonghu; Ma, Wujun; Xia, Xianchun

    2011-03-01

    Polyphenol oxidase (PPO) plays a crucial role in browning reactions in fresh and processed fruits and vegetables, as well as products made from cereal grains. Common wheat (Triticum aestivum L.) has a large genome, representing an interesting system to advance our understanding of plant PPO gene expression, regulation and function. In the present study, we characterized the expression of Ppo-A1, a major PPO gene located on wheat chromosome 2A, using DNA sequencing, semi-quantitative RT-PCR, PPO activity assays and whole-grain staining methods during grain development. The results indicated that the expression of the Ppo-A1b allele was regulated by alternative splicing of pre-mRNAs, resulting from a 191-bp insertion in intron 1 and one C/G SNP in exon 2. Eight mRNA isoforms were identified in developing grains based on alignments between cDNA and genomic DNA sequences. Only the constitutively spliced isoform b encodes a putative full-length PPO protein based on its coding sequence whereas the other seven spliced isoforms, a, c, d, e, f, g and h, have premature termination codons resulting in potential nonsense-mediated mRNA decay. The differences in expression of Ppo-A1a and Ppo-A1b were confirmed by PPO activity assays and whole grain staining, providing direct evidence for the influence of alternative splicing in the coding region of Ppo-A1 on polyphenol oxidase activity in common wheat grains.

  8. AR-V7 and prostate cancer: The watershed for treatment selection?

    PubMed

    Ciccarese, Chiara; Santoni, Matteo; Brunelli, Matteo; Buti, Sebastiano; Modena, Alessandra; Nabissi, Massimo; Artibani, Walter; Martignoni, Guido; Montironi, Rodolfo; Tortora, Giampaolo; Massari, Francesco

    2016-02-01

    The androgen receptor (AR) plays a key role in progression to metastatic castration-resistant prostate cancer (mCRPC). Despite the recent progress in targeting persistent AR activity with the next-generation hormonal therapies (abiraterone acetate and enzalutamide), resistance to these agents limits therapeutic efficacy for many patients. Several explanations for response and/or resistance to abiraterone acetate and enzalutamide are emerging, but growing interest is focusing on importance of AR splice variants (AR-Vs) and in particular of AR-V7. Increasing evidences highlight the concept that variant expression could be used as a potential predictive biomarker and a therapeutic target in advanced prostate cancer. Therefore, understanding the mechanisms of treatment resistance or sensitivity can help to achieve a more effective management of mCRPC, increasing clinical outcomes and representing a promising and engaging area of prostate cancer research.

  9. Calcium Activated K+ Channels in The Electroreceptor of the Skate Confirmed by Cloning. Details of Subunits and Splicing

    PubMed Central

    King, Benjamin L.; Shi, Ling Fang; Kao, Peter; Clusin, William T.

    2015-01-01

    Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K+ channels, first described in l974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intracellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted˜ in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the β subunits (β4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues. PMID:26687710

  10. Peroxisome proliferator-activated receptor gamma in the human pituitary gland: expression and splicing pattern in adenomas versus normal pituitary.

    PubMed

    Occhi, G; Albiger, N; Berlucchi, S; Gardiman, M; Scanarini, M; Scienza, R; Fassina, A; Mantero, F; Scaroni, C

    2007-07-01

    Pituitary adenomas are slow-growing tumours arising within the pituitary gland. If secreting, they give rise to well-known syndromes such as Cushing's disease or acromegaly; when hormonally inactive, they come to clinical attention often with local mass effects or pituitary deficiency. Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor with a key role in fat and glucose metabolism, but also involved in several neoplasia, has recently been detected in pituitary adenomas. In the present study, we evaluated the occurrence and splicing profile of PPARgamma in 43 cases of pituitary adenoma of different subtypes and compared it to 12 normal pituitary glands. By real-time polymerase chain reaction, PPARgamma was expressed as much in adrenocorticotrophic hormone (ACTH)-secreting and ACTH-silent adenomas as in controls, with a moderate underexpression in somatotrophinomas and prolactinomas and overexpression in 54% of nonfunctioning pituitary adenomas (NFPA). There was no apparent qualitative change in the splicing profile of pathological pituitary glands, nor was the presence of specific isoforms with dominant negative effects against PPARgamma detected. Western blotting revealed similar expression levels in the different subgroups of pituitary adenomas and normal glands. Immunohistochemistry confirmed PPARgamma expression in approximately one-half of analysed samples. The intra- and intergroup differences observed in pituitary adenomas may represent new elements in the process of understanding the different clinical responses of Cushing's and Nelson patients to PPARgamma-ligand treatment. Moreover, the higher level of PPARgamma expression detected in the NFPA subgroup may suggest its possible role as a molecular target in these pituitary adenomas, paving the way for investigations on the effectiveness of treatment with thiazolidinediones in such patients.

  11. Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

    PubMed Central

    Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

    2013-01-01

    Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

  12. BRCA1 inhibits AR-mediated proliferation of breast cancer cells through the activation of SIRT1.

    PubMed

    Zhang, Wenwen; Luo, Jiayan; Yang, Fang; Wang, Yucai; Yin, Yongmei; Strom, Anders; Gustafsson, Jan Åke; Guan, Xiaoxiang

    2016-02-23

    Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor protein that functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. The androgen receptor (AR) is expressed in more than 70% of breast cancers and has been implicated in breast cancer pathogenesis. However, little is known about the role of BRCA1 in AR-mediated cell proliferation in human breast cancer. Here, we report that a high expression of AR in breast cancer patients was associated with shorter overall survival (OS) using a tissue microarray with 149 non-metastatic breast cancer patient samples. We reveal that overexpression of BRCA1 significantly inhibited expression of AR through activation of SIRT1 in breast cancer cells. Meanwhile, SIRT1 induction or treatment with a SIRT1 agonist, resveratrol, inhibits AR-stimulated proliferation. Importantly, this mechanism is manifested in breast cancer patient samples and TCGA database, which showed that low SIRT1 gene expression in tumor tissues compared with normal adjacent tissues predicts poor prognosis in patients with breast cancer. Taken together, our findings suggest that BRCA1 attenuates AR-stimulated proliferation of breast cancer cells via SIRT1 mediated pathway.

  13. Understanding splicing regulation through RNA splicing maps.

    PubMed

    Witten, Joshua T; Ule, Jernej

    2011-03-01

    Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

  14. Pharmacology of Modulators of Alternative Splicing

    PubMed Central

    Morris, Jonathan C.; Oltean, Sebastian; Donaldson, Lucy F.

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. PMID:28034912

  15. Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain*

    PubMed Central

    Banuelos, Carmen A.; Tavakoli, Iran; Tien, Amy H.; Caley, Daniel P.; Mawji, Nasrin R.; Li, Zhenzhen; Wang, Jun; Yang, Yu Chi; Imamura, Yusuke; Yan, Luping; Wen, Jian Guo; Andersen, Raymond J.; Sadar, Marianne D.

    2016-01-01

    Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis. PMID:27576691

  16. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability.

    PubMed

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-02-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated.

  17. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability

    PubMed Central

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-01-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated. PMID:24356448

  18. Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo.

    PubMed

    Kwegyir-Afful, Andrew K; Ramalingam, Senthilmurugan; Purushottamachar, Puranik; Ramamurthy, Vidya P; Njar, Vincent C O

    2015-09-29

    Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

  19. Differential activation of human constitutive androstane receptor and its SV23 and SV24 splice variants by rilpivirine and etravirine

    PubMed Central

    Sharma, Devinder; Lau, Aik Jiang; Sherman, Matthew A; Chang, Thomas K H

    2015-01-01

    Background and Purpose Rilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). Experimental Approach Receptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. Key Results Rilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. Conclusion and Implications NNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3. PMID:25363652

  20. The geology and 40Ar/ 39Ar geochronology of magmatic activity and related mineralization in the Nevados del Famatina mining district, La Rioja province, Argentina

    NASA Astrophysics Data System (ADS)

    Losada-Calderón, A. J.; McBride, S. L.; Bloom, M. S.

    1994-01-01

    The Nevados del Famatina mining district (NFMD) is located in La Rioja province, Argentina. This district contains porphyry-style mineralization (Nevados del Famatina) and high sulfidation veins (La Mejicana). The stratigraphic column in the NFMD begins with Cambrian siltstones which were metamorphosed during the Late Ordovician - Early Silurian and intruded by Late Ordovician-Silurian granitic rocks. These units were covered by Upper Paleozoic and Tertiary continental sedimentary rocks which are intercalated with and overlain by dacitic-rhyodacitic porphyritic rocks (Mogote Formation) emplaced during the Pliocene. All these units are covered by Pleistocene sediments and Quaternary alluvial and colluvial deposits. Magmatic activity and related mineralization in the NFMD have been dated by the 40Ar/ 39Ar technique. Step heating studies of orthoclase and biotite phenocrysts from the Mogote Formation in the NFMD suggest that the igneous rocks were emplaced around 5.0±0.3 Ma ago. However, plateau ages of biotite from the outer carapace of the subjacent granodioritic magma chamber and of muscovite from quartz-sericite alteration at both Nevados del Famatina and La Mejicana are around 3.8±0.2 Ma. Emplacement of the shallow stocks is separated from cooling of the outer carapace of the subjacent granodioritic magma chamber to temperatures below 350° C by a time span of approximately 1 Ma. During this interval, a convective hydrothermal system was established proximal to the granodioritic magma chamber, which resulted in porphyry molybdenumcoppergold mineralization adjacent to the igneous rocks and more distal high sulfidation veins located in fault zones.

  1. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  2. Cryptic splice sites and split genes.

    PubMed

    Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M; Tatusova, Tatiana; Dibb, Nick J

    2011-08-01

    We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes.

  3. Cryptic splice sites and split genes

    PubMed Central

    Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M.; Tatusova, Tatiana; Dibb, Nick J.

    2011-01-01

    We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes. PMID:21470962

  4. Titanium spallation cross sections between 30 and 584 MeV and Ar-39 activities on the moon

    NASA Technical Reports Server (NTRS)

    Steinburnn, F.; Fireman, E. L.

    1974-01-01

    The production cross sections of Ar39 for Ti spallation at 45-, 319-, 433-, and 584-MeV proton energies were measured to be 0.37 + or - 0.09, 12.4 + or - 3.7, 9.1 + or - 2.7, and 17.8 + or - 6.2 mb, respectively. Normalized Ar39 production rates and activities are also derived for protons above 40 MeV and for three differential proton spectra of the type approximately E(- alpha). It is concluded that, even for samples of high-Ti content, Ti spallation by solar protons below 200-MeV energy does not contribute significantly to their Ar39 radioactivity.

  5. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    PubMed

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  6. 20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor

    PubMed Central

    Cao, Bo; Liu, Xichun; Li, Jing; Liu, Shuang; Qi, Yanfeng; Xiong, Zhenggang; Zhang, Allen; Wiese, Thomas; Fu, Xueqi; Gu, Jingkai; Rennie, Paul S.; Sartor, Oliver; Lee, Benjamin R.; Ip, Clement; Zhao, Lijuan; Zhang, Haitao; Dong, Yan

    2012-01-01

    As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively-active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer. PMID:22907191

  7. 5'- and 3'-terminal nucleotides in the FGFR2 ISAR splicing element core have overlapping roles in exon IIIb activation and exon IIIc repression.

    PubMed

    Jones, R B; Carstens, R P; Luo, Y; McKeehan, W L

    2001-09-01

    The cell type-specific, mutually-exclusive alternative splicing of the fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is tightly regulated. A sequence termed ISAR (intronic splicing activator and repressor) has been implicated as an important cis regulatory element in both activation of exon IIIb and repression of exon IIIc splicing in epithelial cells. In order to better understand how this single sequence could have dual roles, we transfected minigenes containing a series of 2-bp mutations in the 18 3'-most nucleotides of ISAR that we refer to as the ISAR core. Transfection of cells with dual-exon (IIIb and IIIc) minigenes revealed that mutation of terminal sequences of the core led to decreased exon IIIb inclusion and increased exon IIIc inclusion. Transfection of cells with single-exon IIIb minigenes and single-exon IIIc minigenes revealed that mutation of terminal sequences of the ISAR core led to decreased exon IIIb inclusion and increased exon IIIc inclusion, respectively. Nucleotides of the ISAR core responsible for exon IIIb activation appear to overlap very closely with those required for exon IIIc repression. We describe a model in which ISAR and a 5' intronic sequence known as IAS2 form a stem structure required for simultaneous exon IIIb activation and exon IIIc repression.

  8. Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.

    PubMed

    Lamech, Lilian T; Mallam, Anna L; Lambowitz, Alan M

    2014-12-01

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was "fixed" by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

  9. Alternative splicing of the androgen receptor in polycystic ovary syndrome

    PubMed Central

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C. K.; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-01-01

    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. PMID:25825716

  10. The relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro.

    PubMed

    Rouleau, Mélanie; Roberge, Joannie; Falardeau, Sarah-Ann; Villeneuve, Lyne; Guillemette, Chantal

    2013-04-01

    Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.

  11. CALCULATING SEPARATE MAGNETIC FREE ENERGY ESTIMATES FOR ACTIVE REGIONS PRODUCING MULTIPLE FLARES: NOAA AR11158

    SciTech Connect

    Tarr, Lucas; Longcope, Dana; Millhouse, Margaret

    2013-06-10

    It is well known that photospheric flux emergence is an important process for stressing coronal fields and storing magnetic free energy, which may then be released during a flare. The Helioseismic and Magnetic Imager (HMI) on board the Solar Dynamics Observatory (SDO) captured the entire emergence of NOAA AR 11158. This region emerged as two distinct bipoles, possibly connected underneath the photosphere, yet characterized by different photospheric field evolutions and fluxes. The combined active region complex produced 15 GOES C-class, two M-class, and the X2.2 Valentine's Day Flare during the four days after initial emergence on 2011 February 12. The M and X class flares are of particular interest because they are nonhomologous, involving different subregions of the active region. We use a Magnetic Charge Topology together with the Minimum Current Corona model of the coronal field to model field evolution of the complex. Combining this with observations of flare ribbons in the 1600 A channel of the Atmospheric Imaging Assembly on board SDO, we propose a minimization algorithm for estimating the amount of reconnected flux and resulting drop in magnetic free energy during a flare. For the M6.6, M2.2, and X2.2 flares, we find a flux exchange of 4.2 Multiplication-Sign 10{sup 20} Mx, 2.0 Multiplication-Sign 10{sup 20} Mx, and 21.0 Multiplication-Sign 10{sup 20} Mx, respectively, resulting in free energy drops of 3.89 Multiplication-Sign 10{sup 30} erg, 2.62 Multiplication-Sign 10{sup 30} erg, and 1.68 Multiplication-Sign 10{sup 32} erg.

  12. Alternative splicing of CARMA2/CARD14 transcripts generates protein variants with differential effect on NF-κB activation and endoplasmic reticulum stress-induced cell death.

    PubMed

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-12-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis.

  13. Alternative Splicing of CARMA2/CARD14 Transcripts Generates Protein Variants With Differential Effect on NF-κB Activation and Endoplasmic Reticulum Stress-Induced Cell Death

    PubMed Central

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-01-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis. J. Cell. Physiol. 226: 3121–3131, 2011. © 2011 Wiley Periodicals, Inc. PMID:21302310

  14. BhbZIP60 from Resurrection Plant Boea hygrometrica Is an mRNA Splicing-Activated Endoplasmic Reticulum Stress Regulator Involved in Drought Tolerance

    PubMed Central

    Wang, Bo; Du, Hong; Zhang, Zhennan; Xu, Wenzhong; Deng, Xin

    2017-01-01

    Adverse environmental conditions cause endoplasmic reticulum (ER) stress in plants. To mitigate ER stress damage, ER associated transcription factors and inositol-requiring enzyme-1 (IRE1)-mediated bZIP60 mRNA splicing are activated in plants. A drought-induced gene, encoding the ortholog of AtbZIP60, was identified in the resurrection plant Boea hygrometrica, termed BhbZIP60. In response to ER stress and dehydration, BhbZIP60 mRNA can be spliced to create a frame shift in the C terminus by the excision of 23b segment in a manner of its ortholog in other plants, thus translocating to the nucleus instead of the cytoplasm. The splicing-activated BhbZIP60 (BhbZIP60S) could function in the same way as its Arabidopsis ortholog by restoring the molecular phenotype of the mutant atbzip60. When overexpressed in Arabidopsis, BhbZIP60S provided transgenic plants with enhanced tolerance to drought, tunicamycin and mannitol stresses with upregulation of the expressions of ER quality control (QC) genes (BiP2, BiP3, CNX1, and sPDI) and abscisic acid (ABA) responsive genes (RD29A, RAB18, and RD17). Furthermore, in the yeast one-hybrid system, BhbZIP60S was capable of interacting with ER stress responsive elements (ERSE and ERSE-II) that exist in the promoters of known ER-QC genes, but not binding to ABA responsive cis-elements (ABREs). Our results demonstrated that drought-induced BhbZIP60 may have a function in drought tolerance via the splicing-activated BhbZIP60S to mediate ER-QC by direct binding to the promoters of ER-QC genes. This study evidently demonstrates the involvement of ER-QC in the drought tolerance of Arabidopsis and the desiccation tolerance of the resurrection plant B. hygrometrica. PMID:28286511

  15. BhbZIP60 from Resurrection Plant Boea hygrometrica Is an mRNA Splicing-Activated Endoplasmic Reticulum Stress Regulator Involved in Drought Tolerance.

    PubMed

    Wang, Bo; Du, Hong; Zhang, Zhennan; Xu, Wenzhong; Deng, Xin

    2017-01-01

    Adverse environmental conditions cause endoplasmic reticulum (ER) stress in plants. To mitigate ER stress damage, ER associated transcription factors and inositol-requiring enzyme-1 (IRE1)-mediated bZIP60 mRNA splicing are activated in plants. A drought-induced gene, encoding the ortholog of AtbZIP60, was identified in the resurrection plant Boea hygrometrica, termed BhbZIP60. In response to ER stress and dehydration, BhbZIP60 mRNA can be spliced to create a frame shift in the C terminus by the excision of 23b segment in a manner of its ortholog in other plants, thus translocating to the nucleus instead of the cytoplasm. The splicing-activated BhbZIP60 (BhbZIP60S) could function in the same way as its Arabidopsis ortholog by restoring the molecular phenotype of the mutant atbzip60. When overexpressed in Arabidopsis, BhbZIP60S provided transgenic plants with enhanced tolerance to drought, tunicamycin and mannitol stresses with upregulation of the expressions of ER quality control (QC) genes (BiP2, BiP3, CNX1, and sPDI) and abscisic acid (ABA) responsive genes (RD29A, RAB18, and RD17). Furthermore, in the yeast one-hybrid system, BhbZIP60S was capable of interacting with ER stress responsive elements (ERSE and ERSE-II) that exist in the promoters of known ER-QC genes, but not binding to ABA responsive cis-elements (ABREs). Our results demonstrated that drought-induced BhbZIP60 may have a function in drought tolerance via the splicing-activated BhbZIP60S to mediate ER-QC by direct binding to the promoters of ER-QC genes. This study evidently demonstrates the involvement of ER-QC in the drought tolerance of Arabidopsis and the desiccation tolerance of the resurrection plant B. hygrometrica.

  16. Tectonic events, continental intraplate volcanism, and mantle plume activity in northern Arabia: Constraints from geochemistry and Ar-Ar dating of Syrian lavas

    NASA Astrophysics Data System (ADS)

    Krienitz, M.-S.; Haase, K. M.; Mezger, K.; van den Bogaard, P.; Thiemann, V.; Shaikh-Mashail, M. A.

    2009-04-01

    New 40Ar/39Ar ages combined with chemical and Sr, Nd, and Pb isotope data for volcanic rocks from Syria along with published data of Syrian and Arabian lavas constrain the spatiotemporal evolution of volcanism, melting regime, and magmatic sources contributing to the volcanic activity in northern Arabia. Several volcanic phases occurred in different parts of Syria in the last 20 Ma that partly correlate with different tectonic events like displacements along the Dead Sea Fault system or slab break-off beneath the Bitlis suture zone, although the large volume of magmas and their composition suggest that hot mantle material caused volcanism. Low Ce/Pb (<20), Nb/Th (<10), and Sr, Nd, and Pb isotope variations of Syrian lavas indicate the role of crustal contamination in magma genesis, and contamination of magmas with up to 30% of continental crustal material can explain their 87Sr/86Sr. Fractionation-corrected major element compositions and REE ratios of uncontaminated lavas suggest a pressure-controlled melting regime in western Arabia that varies from shallow and high-degree melt formation in the south to increasingly deeper regions and lower extents of the beginning melting process northward. Temperature estimates of calculated primary, crustally uncontaminated Arabian lavas indicate their formation at elevated mantle temperatures (Texcess ˜ 100-200°C) being characteristic for their generation in a plume mantle region. The Sr, Nd, and Pb isotope systematic of crustally uncontaminated Syrian lavas reveal a sublithospheric and a mantle plume source involvement in their formation, whereas a (hydrous) lithospheric origin of lavas can be excluded on the basis of negative correlations between Ba/La and K/La. The characteristically high 206Pb/204Pb (˜19.5) of the mantle plume source can be explained by material entrainment associated with the Afar mantle plume. The Syrian volcanic rocks are generally younger than lavas from the southern Afro-Arabian region, indicating

  17. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

    SciTech Connect

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard; Bies, Juraj; Wolff, Linda; Mougel, Marylene . E-mail: mmougel@univ-montp1.fr

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

  18. Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells

    PubMed Central

    Ajith, Sandya; Gazzara, Matthew R.; Cole, Brian S.; Shankarling, Ganesh; Martinez, Nicole M.; Mallory, Michael J.; Lynch, Kristen W.

    2016-01-01

    ABSTRACT CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts. PMID:27096301

  19. Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB.

    PubMed

    Mukai, Satomi; Kitazawa, Riko; Ishii, Junko; Kondo, Takeshi; Hakozaki, Akihiro; Horiuchi, Keisuke; Haraguch, Ryuma; Mori, Kiyoshi; Kitazawa, Sohei

    2011-04-01

    Receptor activator of NF-κB (RANK) is a member of the tumor necrosis factor receptor (TNFR) family expressed in osteoclast precursors, and RANK-RANK ligand (RANKL) signaling is a key system for differentiation, activation and survival of osteoclasts. Here, we report the identification of a novel alternative splicing variant of mouse RANK gene (vRANK) that contains a new intervening exon between exon 1 and exon 2 of mouse full-length RANK (fRANK) mRNA. Since this novel exon contains the stop codon, vRANK encodes truncated amino acids that have a portion of the signal peptide of fRANK and an additional 19 amino acids that show no homology to previously reported domains. By transient transfection studies with vRANK-GFP and -Flag expressing constructs, vRANK was found localized mostly in the cytoplasm and partly in the cell membrane, but was not secreted into the culture supernatant. Under the stimulation of various factors, the expression of vRANK mRNA was almost parallel to that of fRANK in RAW264.7 cells not treated with M-CSF. Overexpression of vRANK, on the other hand, decreased TRACP (a marker of osteoclasts) mRNA expression as well as the number of TRACP-positive multinucleated giant cells. While the mRNA expression levels of NFATc1 (a master transcriptional factor of the osteoclast differentiation program) were not affected, apoptotic cells increased significantly in vRAN K-transfected cells treated with sRANKL. Taken together, these results suggest that vRANK is a novel osteoclast suppressor that reduces the number of RANKL-induced mature osteoclasts mainly by negating the anti-apoptotic effect of RANKL.

  20. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    PubMed Central

    Hall, Vanessa J.; Lindblad, Maiken M.; Jakobsen, Jannik E.; Gunnarsson, Anders; Schmidt, Mette; Rasmussen, Mikkel A.; Volke, Daniela; Zuchner, Thole; Hyttel, Poul

    2015-01-01

    ABSTRACT Animal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation. PMID:26398935

  1. TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity

    SciTech Connect

    Na, Lei; Tang, Yan-Dong; Liu, Jian-Dong; Yu, Chang-Qing; Sun, Liu-Ke; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun; Zhou, Jian-Hua

    2014-04-04

    Highlights: • TRIMe7-CypA expresses in rhesus and pig-tailed, but not long-tailed macaques. • TRIMe7-CypA does not show the restriction to a HIV-GFP report virus in vitro. • It acts as a negative modulator to TRIM5α likely by competitive inhibition. - Abstract: The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition.

  2. MED1 mediates androgen receptor splice variant induced gene expression in the absence of ligand

    PubMed Central

    Liu, Gang; Sprenger, Cynthia; Wu, Pin-Jou; Sun, Shihua; Uo, Takuma; Haugk, Kathleen; Epilepsia, Kathryn Soriano; Plymate, Stephen

    2015-01-01

    The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation has not been defined. A distinct transcriptome enriched with cell cycle genes, e.g. UBE2C, has been associated with AR-Vs, which indicates the possibility of an altered transcriptional mechanism when compared to full-length wild-type AR (ARfl). Importantly, a recent study reported the critical role of p-MED1 in enhancing UBE2C expression through a locus looping pattern, which only occurs in CRPC but not in androgen-dependent prostate cancer (ADPC). To investigate the potential correlation between AR-V and MED1, in the present study we performed protein co-immunoprecipitation, chromatin immunoprecipitation, and cell proliferation assays and found that MED1 is necessary for ARv567es induced UBE2C up-regulation and subsequent prostate cancer cell growth. Furthermore, p-MED1 is bound to ARv567es independent of full-length AR; p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of ARv567es. Our data indicate that p-MED1 serves as a key mediator in ARv567es induced gene expression and suggests a mechanism by which AR-Vs promote the development and progression of CRPC. PMID:25481872

  3. Splicing Factor Prp8 Interacts With NESAR and Regulates Androgen Receptor in Prostate Cancer Cells

    PubMed Central

    Wang, Dan; Nguyen, Minh M.; Masoodi, Khalid Z.; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A.; Dhir, Rajiv

    2015-01-01

    Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NESAR) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NESAR, we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NESAR export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NESAR function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NESAR. Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NESAR and regulates AR function in prostate cancer cells. PMID:26371515

  4. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  5. USDA-ARS extension activities in medical, veterinary and urban entomology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Within the USDA Agricultural Research Service (USDA-ARS), National Program 104 conducts research on veterinary, medical, and urban entomology. The goal of this program is to develop more effective methods of preventing or suppressing insects, ticks, and mites that affect animal and human well-being....

  6. Systematic identification and analysis of exonic splicing silencers.

    PubMed

    Wang, Zefeng; Rolish, Michael E; Yeo, Gene; Tung, Vivian; Mawson, Matthew; Burge, Christopher B

    2004-12-17

    Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

  7. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways.

    PubMed

    Niu, Dong-Dong; Liu, Hong-Xia; Jiang, Chun-Hao; Wang, Yun-Peng; Wang, Qing-Ya; Jin, Hai-Ling; Guo, Jian-Hua

    2011-05-01

    Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.

  8. Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron

    PubMed Central

    Monachello, Dario; Michel, François; Costa, Maria

    2016-01-01

    When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled. PMID:26769855

  9. Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron.

    PubMed

    Monachello, Dario; Michel, François; Costa, Maria

    2016-03-01

    When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled.

  10. Multiple links between transcription and splicing.

    PubMed

    Kornblihtt, Alberto R; de la Mata, Manuel; Fededa, Juan Pablo; Munoz, Manuel J; Nogues, Guadalupe

    2004-10-01

    Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

  11. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  12. Analysis of mitogen-activated protein kinase activation by naturally occurring splice variants of TrkC, the receptor for neurotrophin-3.

    PubMed

    Gunn-Moore, F J; Williams, A G; Tavaré, J M

    1997-02-15

    TrkC is a receptor tyrosine kinase that binds neurotrophin-3 (NT-3) with high affinity. A number of naturally occurring splice variants of TrkC exist, including one (TrkC kil4) with a 14 amino acid insertion between subdomains VII and VIII of the tyrosine kinase domain. This kinase insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block the ability of TrkC to form a high-affinity complex with Shc and phospholipase C gamma (PLC gamma) and the activation of PtdIns 3-kinase, and attenuates the sustained activation of mitogen-activated protein kinase (MAPK). In the current study we set out to determine whether the attenuation of the activation of MAPK by the insert was the result of the inability of TrkC to activate the Shc-Ras pathway, PtdIns 3-kinase activation, PLC gamma activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PLC gamma and PtdIns 3-kinase in the activation of MAPK by TrkC. The introduction of the 14 amino acid kinase insert appeared to slow the kinetics of NT-3-stimulated Shc phosphorylation and Shc-Grb2 association and reduce their magnitude; an effect which was associated with a delayed, and only transient, activation of MAPK. Taken together, our data suggest that the apparent defect in MAPK activation caused by the kinase insert may result predominantly from an inhibition of high-affinity Shc binding, although a role for PLC gamma and PtdIns 3-kinase cannot be completely excluded.

  13. The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

    PubMed Central

    Schmauss, Claudia; Zimnisky, Ross; Mehta, Mukti; Shapiro, Lauren P.

    2010-01-01

    The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes. PMID:20651031

  14. {sup 40}Ar/{sup 39}Ar laser fusion and K-Ar ages from Lathrop Wells, Nevada, and Cima, California: The age of the latest volcanic activity in the Yucca Mountain area

    SciTech Connect

    Turrin, B.D. |; Champion, D.E.

    1991-05-01

    K-Ar and {sup 40}Ar/{sup 39}Ar ages from the Lathrop Wells volcanic center, Nevada, and from the Cima volcanic field, California, indicate that the recently reported 20-ka age estimate for the Lathrop Wells volcanic center is incorrect. Instead, an age of 119 {plus_minus} 11 to 141 {plus_minus} 10 ka is indicated for the Lathrop Wells volcanic center. This age corrected is concordant with the ages determined by two independent isotopic geochronometric techniques and with the stratigraphy of surficial deposits in the Yucca Mountain region. In addition, paleomagnetic data and radiometric age data indicate only two volcanic events at the Lathrop Wells volcanic center that are probably closely linked in time, not as many as five as recently reported. 32 refs., 2 figs., 2 tabs.

  15. 40Ar/39Ar laser fusion and K-Ar ages from Lathrop Wells, Nevada, and Cima, California. The age of the latest volcanic activity in the Yucca Mountain area

    USGS Publications Warehouse

    Turrin, Brent D.; Champion, Duane E.; ,

    1991-01-01

    K-Ar and 40Ar/39Ar ages from the Lathrop Wells volcanic center, Nevada, and from the Cima volcanic field, California, indicate that the recently reported 20-ka age estimate for the Lathrop Wells volcanic center is incorrect. Instead an age of 119??11 to 141??10 ka is indicated for the Lathrop Wells volcanic center. This age corrected is concordant with the ages determined by two independent isotopic geochronometric techniques and with the stratigraphy of surficial deposits in the Yucca Mountain region. In addition, paleomagnetic data and radiometric age data indicate only two volcanic events at the Lathrop Wells volcanic center that are probably closely linked in time, not as many as five as recently reported.

  16. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

    PubMed Central

    Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

    2016-01-01

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

  17. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

    PubMed

    Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

    2016-12-27

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

  18. Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3′ splice-site selection. PMID:9858550

  19. Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3β inhibitor, promotes prostate cancer growth in soft tissue and bone

    PubMed Central

    Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; McCauley, Laurie K.; Keller, Evan T.

    2013-01-01

    Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling through inducing β-catenin degradation. Therefore, AR79, an inhibitor of GSK3β, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, β-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and β-catenin status, proliferation (with β-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-β-catenin expression and increased total and nuclear β-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation. PMID:24088787

  20. A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

    SciTech Connect

    Hashimoto, Koshi; Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi; Monden, Tsuyoshi; Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo

    2009-12-25

    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  1. Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing.

    PubMed

    van Kuilenburg, André B P; Meijer, Judith; Maurer, Dirk; Dobritzsch, Doreen; Meinsma, Rutger; Los, Maartje; Knegt, Lia C; Zoetekouw, Lida; Jansen, Rob L H; Dezentjé, Vincent; van Huis-Tanja, Lieke H; van Kampen, Roel J W; Hertz, Jens Michael; Hennekam, Raoul C M

    2017-03-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

  2. Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain

    PubMed Central

    Uo, T; Dvinge, H; Sprenger, C C; Bradley, R K; Nelson, P S; Plymate, S R

    2017-01-01

    The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4–8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD. PMID:27694897

  3. Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction.

    PubMed

    Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J; Bose, Mahuya; Whittal, Randy M; Bose, Himangshu S

    2015-01-30

    Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221-229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis.

  4. The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer.

    PubMed

    Palhais, Bruno; Dembic, Maja; Sabaratnam, Rugivan; Nielsen, Kira S; Doktor, Thomas Koed; Bruun, Gitte Hoffmann; Andresen, Brage Storstein

    2016-11-01

    Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5'splice site (5'ss) that prevents pseudoexon inclusion. The c.639+919 G>A mutation disrupts this ESS allowing U1 snRNP recognition of the 5'ss. We show that the wild-type GLA 5'ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.

  5. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM.

    PubMed

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-11-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

  6. Zinc-induced modulation of SRSF6 activity alters Bim splicing to promote generation of the most potent apoptotic isoform BimS.

    PubMed

    Hara, Hirokazu; Takeda, Tatsuya; Yamamoto, Nozomi; Furuya, Keisuke; Hirose, Kazuya; Kamiya, Tetsuro; Adachi, Tetsuo

    2013-07-01

    Bim is a member of the pro-apoptotic BH3-only Bcl-2 family of proteins. Bim gene undergoes alternative splicing to produce three predominant splicing variants (BimEL, BimL and BimS). The smallest variant BimS is the most potent inducer of apoptosis. Zinc (Zn(2+)) has been reported to stimulate apoptosis in various cell types. In this study, we examined whether Zn(2+) affects the expression of Bim in human neuroblastoma SH-SY5Y cells. Zn(2+) triggered alterations in Bim splicing and induced preferential generation of BimS, but not BimEL and BimL, in a dose- and time-dependent manner. Other metals (cadmium, cobalt and copper) and stresses (oxidative, endoplasmic reticulum and genotoxic stresses) had little or no effect on the expression of BimS. To address the mechanism of Zn(2+)-induced preferential generation of BimS, which lacks exon 4, we developed a Bim mini-gene construct. Deletion analysis using the Bim mini-gene revealed that predicted binding sites of the SR protein SRSF6, also known as SRp55, are located in the intronic region adjacent to exon 4. We also found that mutations in the predicted SRSF6-binding sites abolished generation of BimS mRNA from the mutated Bim mini-gene. In addition, a UV cross-linking assay followed by Western blotting showed that SRSF6 directly bound to the predicted binding site and Zn(2+) suppressed this binding. Moreover, Zn(2+) stimulated SRSF6 hyper-phosphorylation. TG003, a cdc2-like kinase inhibitor, partially prevented Zn(2+)-induced generation of BimS and SRSF6 hyper-phosphorylation. Taken together, our findings suggest that Zn(2+) inhibits the activity of SRSF6 and promotes elimination of exon 4, leading to preferential generation of BimS.

  7. Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments (CLIA) Laboratory Setting.

    PubMed

    Lokhandwala, Parvez M; Riel, Stacy L; Haley, Lisa; Lu, Changxue; Chen, Yan; Silberstein, John; Zhu, Yezi; Zheng, Gang; Lin, Ming-Tseh; Gocke, Christopher D; Partin, Alan W; Antonarakis, Emmanuel S; Luo, Jun; Eshleman, James R

    2017-01-01

    Patients with castration-resistant prostate cancer (CRPC) often are treated with drugs that target the androgen receptor (AR) ligand-binding domain. Constitutively active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells, may be associated with resistance to these agents. We validated an AR-V7 assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Circulating tumor cells were isolated, and mRNA was reverse-transcribed into cDNA. Real-time quantitative PCR amplification of reference transcripts (beta-actin and glyceraldehyde-3-phosphate dehydrogenase), prostate-specific transcripts (prostate-specific membrane antigen, prostate-specific antigen, and AR-full length), and AR-V7 was performed. Specimens for validation included an AR-V7 expressing prostate cancer (LNCaP95), 38 peripheral blood controls, and 21 blood samples from CRPC patients. The assay detected as few as five LNCaP95 cells spiked into peripheral blood, showing high analytical sensitivity. Multiple inter-run and intrarun replicates of LNCaP95 cell line experiments yielded similar cycle threshold values for all genes, showing high analytical precision (AR-V7 cycle threshold CV of 0.67%). All 38 healthy control samples were negative for AR-V7, showing high diagnostic specificity (100%). The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy). This first validated clinical assay detects the AR-V7 with high analytical sensitivity, precision, specificity, and accuracy.

  8. A novel TMEM16A splice variant lacking the dimerization domain contributes to calcium-activated chloride secretion in human sweat gland epithelial cells.

    PubMed

    Ertongur-Fauth, Torsten; Hochheimer, Andreas; Buescher, Joerg Martin; Rapprich, Stefan; Krohn, Michael

    2014-11-01

    Sweating is an important physiological process to regulate body temperature in humans, and various disorders are associated with dysregulated sweat formation. Primary sweat secretion in human eccrine sweat glands involves Ca(2+) -activated Cl(-) channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia; however, their molecular identity in sweat glands remained elusive. Here, we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(acΔe3) lacking the TMEM16A-dimerization domain. Chloride flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca(2+) -dependent Cl(-) secretion in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(acΔe3) is forming a functional CaCC, with basal and Ca(2+) -activated Cl(-) permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl(-) secretion providing opportunities to develop sweat gland-specific therapeutics for treatment of sweating disorders.

  9. A K+ channel splice variant common in human heart lacks a C-terminal domain required for expression of rapidly activating delayed rectifier current.

    PubMed

    Kupershmidt, S; Snyders, D J; Raes, A; Roden, D M

    1998-10-16

    We have cloned HERG USO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is approximately 2-fold more abundant than that for HERG1 (the originally described cDNA). After transfection of HERG USO in Ltk- cells, no current was observed. However, coexpression of HERG USO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO, HERGDeltaC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGDeltaC, indicating that the C terminus of HERG1 includes a domain (splice site or the C-terminal. We suggest that the amplitude and gating of cardiac IKr depends on expression of both HERG1 and HERG USO.

  10. Novel Stably Transfected Human Reporter Cell Line AIZ-AR as a Tool for an Assessment of Human Androgen Receptor Transcriptional Activity

    PubMed Central

    Bartonkova, Iveta; Novotna, Aneta; Dvorak, Zdenek

    2015-01-01

    Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications. PMID:25811655

  11. Bacillus cereus AR156 activates PAMP-triggered immunity and induces a systemic acquired resistance through a NPR1-and SA-dependent signaling pathway.

    PubMed

    Niu, Dongdong; Wang, Xiujuan; Wang, Yanru; Song, Xiaoou; Wang, Jiansheng; Guo, Jianhua; Zhao, Hongwei

    2016-01-01

    Induced resistance responses play a potent role in plant defense system against pathogen attack. Bacillus cereus AR156 is a plant growth promoting rhizobacterium (PGPR) that installs induced systemic resistance (ISR) to Pseudomonas syringae pv. tomato (Pst) in Arabidopsis. Here, we show that AR156 leaf infiltration enhances disease resistance in Arabidopsis through the activation of a systemic acquired resistance (SAR). PR1 protein expression and reactive oxygen species (ROS) burst are strongly induced in plants treated with AR156 and inoculated with Pst than that in plants inoculated with Pst only. Moreover, AR156 can trigger SAR in jar1 or ein2 mutants, but not in the NahG transgenic and NPR1 mutant plants. Our results indicate that AR156-induced SAR depends on SA-signaling pathway and NPR1, but not JA and ET. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1 gene expression, which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). Altogether, our results indicate that AR156 can induce SAR by the SA-signaling pathways in an NPR1-dependent manner and involves multiple PTI components.

  12. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

    PubMed

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-08-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

  13. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells

    PubMed Central

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-01-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway. PMID:27446405

  14. α1 Soluble Guanylyl Cyclase (sGC) Splice Forms as Potential Regulators of Human sGC Activity*S⃞

    PubMed Central

    Sharina, Iraida G.; Jelen, Filip; Bogatenkova, Elena P.; Thomas, Anthony; Martin, Emil; Murad, Ferid

    2008-01-01

    Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one α- and one β-subunit. The α1/β1 sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new α1 sGC protein variants generated by alternative splicing. The 363 residue N1-α1 sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-α1 sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-α1 sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-α1, N2-α1 sGC mRNA levels together with RT-PCR analysis for C-α1 sGC demonstrated that the expression of the α1 sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-α1 sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the α1/β1 heterodimer. The C-α1 sGC variant heterodimerizes with the β1 subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-α1 band did not change in response to ODQ treatments, while the level of 83 kDa α1 band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues. PMID:18381288

  15. Splicing plastic optical fibers

    NASA Astrophysics Data System (ADS)

    Carson, Susan D.; Salazar, Roberto A.

    1991-12-01

    Polymethylmethacrylate (PMMA) plastic optical fiber (500 micrometers diameter, fluoropolymer cladding) has been spliced using a fused silica sleeve and a variety of solvent/PMMA solutions as adhesives. Mechanical splicing using index matching fluid has also been investigated. To ensure good bonding and minimize scattering, fiber ends are polished prior to application of adhesive. Using an LED ((lambda) max approximately 640 nm), losses are routinely less than 1.0 dB/splice, and some adhesive formulations have exhibited losses as low as 0.2 dB/splice. Five-meter fibers with as many as ten splices/fiber have been monitored over a period of several months. No fiber has exhibited an increase in optical loss with time.

  16. Deoxycholic acid inhibited proliferation and induced apoptosis and necrosis by regulating the activity of transcription factors in rat pancreatic acinar cell line AR42J.

    PubMed

    Zhang, Guixin; Zhang, Jingwen; Shang, Dong; Qi, Bing; Chen, Hailong

    2015-09-01

    The objective of this study is to investigate the effect of deoxycholic acid (DCA) on rat pancreatic acinar cell line AR42J and the functional mechanisms of DCA on AR42J cells. AR42J cells were treated with various concentrations of DCA for 24 h and also treated with 0.4 mmol/L DCA for multiple times, and then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect the AR42J cell survival rate. Flow cytometric was used to detect the cell apoptosis and necrosis in AR42J cells treated with 0.4 mmol/L and 0.8 mmol/L DCA. The cells treated with phosphate buffer saline (PBS) were served as control. In addition, the DNA-binding activity assays of transcription factors (TFs) in nuclear proteins of cells treated with DCA were determined using Panomics Procarta Transcription Factor Assay Kit. The relative survival rates were markedly decreased (P < 0.05) in a dose- and time-dependent manner. Compared with control group, the cell apoptosis and necrosis ratio were both significantly elevated in 0.4 mmol/L DCA and 0.8 mmol/L DCA groups (P < 0.01). A significant increase (P < 0.05) in the activity of transcription factor 2 (ATF2), interferon-stimulated response element (ISRE), NKX-2.5, androgen receptor (AR), p53, and hypoxia-inducible factor-1 (HIF-1) was observed, and the activity of peroxisome proliferator-activated receptor (PPAR), activator protein 1 (AP1), and E2F1 was reduced (P < 0.05). In conclusion, DCA inhibited proliferation and induced apoptosis and necrosis in AR42J cells. The expression changes of related genes regulated by TFs might be the molecular mechanism of AR42J cell injury.

  17. Multiple splicing defects in an intronic false exon.

    PubMed

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  18. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  19. Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

    SciTech Connect

    Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de; Brinkmann, A.O.; Degenhart, H.J.; Trapman, J. )

    1994-04-01

    The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.

  20. Assessing the volcanic hazard for Rome: 40Ar/39Ar and In-SAR constraints on the most recent eruptive activity and present-day uplift at Colli Albani Volcanic District

    NASA Astrophysics Data System (ADS)

    Marra, F.; Gaeta, M.; Giaccio, B.; Jicha, B. R.; Palladino, D. M.; Polcari, M.; Sottili, G.; Taddeucci, J.; Florindo, F.; Stramondo, S.

    2016-07-01

    We present new 40Ar/39Ar data which allow us to refine the recurrence time for the most recent eruptive activity occurred at Colli Albani Volcanic District (CAVD) and constrain its geographic area. Time elapsed since the last eruption (36 kyr) overruns the recurrence time (31 kyr) in the last 100 kyr. New interferometric synthetic aperture radar data, covering the years 1993-2010, reveal ongoing inflation with maximum uplift rates (>2 mm/yr) in the area hosting the most recent (<200 ka) vents, suggesting that the observed uplift might be caused by magma injection within the youngest plumbing system. Finally, we frame the present deformation within the structural pattern of the area of Rome, characterized by 50 m of regional uplift since 200 ka and by geologic evidence for a recent (<2000 years) switch of the local stress-field, highlighting that the precursors of a new phase of volcanic activity are likely occurring at the CAVD.

  1. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

    PubMed Central

    Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

    2007-01-01

    Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

  2. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    PubMed Central

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  3. The activation energy for nanocrystalline diamond films deposited from an Ar/H2/CH4 hot-filament reactor.

    PubMed

    Barbosa, D C; Melo, L L; Trava-Airoldi, V J; Corat, E J

    2009-06-01

    In this work we have investigated the effect of substrate temperature on the growth rate and properties of nanocrystalline diamond thin films deposited by hot filament chemical vapor deposition (HFCVD). Mixtures of 0.5 vol% CH4 and 25 vol% H2 balanced with Ar at a pressure of 50 Torr and typical deposition time of 12 h. We present the measurement of the activation energy by accurately controlling the substrate temperature independently of other CVD parameters. Growth rates have been measured in the temperature range from 550 to 800 degrees C. Characterization techniques have involved Raman spectroscopy, high resolution X-ray difractometry and scanning electron microscopy. We also present a comparison with most activation energy for micro and nanocrystalline diamond determinations in the literature and propose that there is a common trend in most observations. The result obtained can be an evidence that the growth mechanism of NCD in HFCVD reactors is very similar to MCD growth.

  4. The influence of Argonaute proteins on alternative RNA splicing.

    PubMed

    Batsché, Eric; Ameyar-Zazoua, Maya

    2015-01-01

    Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO.

  5. RNA-splicing endonuclease structure and function.

    PubMed

    Calvin, K; Li, H

    2008-04-01

    The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes.

  6. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR

  7. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

    PubMed Central

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR

  8. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

  9. The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

    PubMed

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

    The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

  10. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome.

    PubMed

    Finley, Jahahreeh

    2015-09-01

    Although the use of antiretroviral therapy (ART) has proven highly effective in controlling and suppressing HIV-1 replication, the persistence of latent but replication-competent proviruses in a small subset of CD4(+) memory T cells presents significant challenges to viral eradication from infected individuals. Attempts to eliminate latent reservoirs are epitomized by the 'shock and kill' approach, a strategy involving the combinatorial usage of compounds that influence epigenetic modulation and initiation of proviral transcription. However, efficient regulation of viral pre-mRNA splicing through manipulation of host cell splicing machinery is also indispensible for HIV-1 replication. Interestingly, aberrant alternative splicing of the LMNA gene via the usage of a cryptic splice site has been shown to be the cause of most cases of Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic condition characterized by an accelerated aging phenotype due to the accumulation of a truncated form of lamin A known as progerin. Recent evidence has shown that inhibition of the splicing factors ASF/SF2 (or SRSF1) and SRp55 (or SRSF6) leads to a reduction or an increase in progerin at both the mRNA and protein levels, respectively, thus altering the LMNA pre-mRNA splicing ratio. It is also well-established that during the latter stages of HIV-1 infection, an increase in the production and nuclear export of unspliced viral mRNA is indispensible for efficient HIV-1 replication and that the presence of ASF/SF2 leads to excessive viral pre-mRNA splicing and a reduction of unspliced mRNA, while the presence of SRp55 inhibits viral pre-mRNA splicing and aids in the generation and translation of unspliced HIV-1 mRNAs. The splicing-factor associated protein and putative mitochondrial chaperone p32 has also been shown to inhibit ASF/SF2, increase unspliced HIV-1 viral mRNA, and enhance mitochondrial DNA replication and oxidative phosphorylation. It is our hypothesis that activation of

  11. Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, K(Ca)3.1, in immune function of lymphoid cells.

    PubMed

    Ohya, Susumu; Niwa, Satomi; Yanagi, Ayano; Fukuyo, Yuka; Yamamura, Hisao; Imaizumi, Yuji

    2011-05-13

    The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

  12. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  13. Visualizing group II intron catalysis through the stages of splicing

    PubMed Central

    Marcia, Marco; Pyle, Anna Marie

    2012-01-01

    SUMMARY Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report fourteen crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing, in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, adopting a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome. PMID:23101623

  14. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

  15. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  16. RNA helicases in splicing.

    PubMed

    Cordin, Olivier; Beggs, Jean D

    2013-01-01

    In eukaryotic cells, introns are spliced from pre-mRNAs by the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. There is evidence that the various helicases are functionally and physically connected with each other and with many other factors in the spliceosome. Understanding the dynamics of those interactions is essential to comprehend the mechanism and regulation of normal as well as of pathological splicing. This review focuses on recent advances in the characterization of the splicing helicases and their interactions, and highlights the deep integration of splicing helicases in global mRNP biogenesis pathways.

  17. Larvicidal and Biting Deterrent Activity of Essential Oils of Curcuma longa, Ar-turmerone, and Curcuminoids Against Aedes aegypti and Anopheles quadrimaculatus (Culicidae: Diptera).

    PubMed

    Ali, Abbas; Wang, Yan-Hong; Khan, Ikhlas A

    2015-09-01

    Essential oils and extract of Curcuma longa, ar-turmerone, and curcuminoids were evaluated for their larvicidal and deterrent activity against mosquitoes. Ar-turmerone and curcuminoids constituted 36.9, 24.9 and 50.6% of rhizome oil, leaf oil, and rhizome extract, respectively. Ar-turmerone was the major compound of the rhizome oil (36.9%) and leaf oil (24.9%). The ethanolic extract had 15.4% ar-turmerone with 6.6% bisdesmethoxycurcumin, 6.1% desmethoxycurcumin, and 22.6% curcumin. In in vitro studies, essential oils of the leaf (biting deterrence index [BDI] = 0.98), rhizome (BDI = 0.98), and rhizome ethanolic extract (BDI = 0.96) at 10 µg/cm(2) showed biting deterrent activity similar to DEET at 25 nmol/cm(2) against Aedes aegypti L. Among the pure compounds, ar-turmerone (BDI = 1.15) showed the biting deterrent activity higher than DEET at 25 nmol/cm(2) whereas the activity of other compounds was lower than DEET. In Anopheles quadrimaculatus Say, only ar-turmerone showed deterrent activity similar to DEET. In dose-response bioassay, ar-turmerone showed significantly higher biting deterrence than DEET at all the dosages. Ar-turmerone, at 15 nmol/cm(2), showed activity similar to DEET at 25 nmol/cm(2) and activity at 5 nmol/cm(2) was similar to DEET at 20 and 15 nmol/cm(2). Leaf essential oil with LC(50) values of 1.8 and 8.9 ppm against larvae of An. quadrimaculatus and Ae. aegypti, respectively, showed highest toxicity followed by rhizome oil and ethanolic extract. Among the pure compounds, ar-turmerone with LC(50) values of 2.8 and 2.5 ppm against larvae of An. quadrimaculatus and Ae. aegypti, respectively, was most toxic followed by bisdesmethoxycurcumin, curcumin, and desmethoxycurcumin.

  18. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    SciTech Connect

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

  19. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    DOE PAGES

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; ...

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to themore » adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.« less

  20. Insights from Zebrafish and Mouse Models on the Activity and Safety of Ar-Turmerone as a Potential Drug Candidate for the Treatment of Epilepsy

    PubMed Central

    Orellana-Paucar, Adriana Monserrath; Afrikanova, Tatiana; Thomas, Joice; Aibuldinov, Yelaman K.; Dehaen, Wim; de Witte, Peter A. M.; Esguerra, Camila V.

    2013-01-01

    In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application. PMID:24349101

  1. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  2. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

    PubMed Central

    Bergsma, Atze J; in ‘t Groen, Stijn LM; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, WWM Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  3. Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R.

    PubMed

    Izquierdo, José M

    2010-12-01

    T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

  4. Validation of the Soil Moisture Active Passive mission using USDA-ARS experimental watersheds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The calibration and validation program of the Soil Moisture Active Passive mission (SMAP) relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The USDA Agricultural Research Service operates several experimental watersheds wh...

  5. Initial validation of the Soil Moisture Active Passive mission using USDA-ARS watersheds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Soil Moisture Active Passive (SMAP) Mission was launched in January 2015 to measure global surface soil moisture. The calibration and validation program of SMAP relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The U...

  6. The Role of the Kink Instability of a Long-Lived Active Region AR 9604

    NASA Astrophysics Data System (ADS)

    Tian, Lirong; Liu, Yang; Yang, Jing; Alexander, David

    2005-07-01

    We have traced the long-term evolution of a non-Hale active region composed of NOAA 9604 9632 9672 9704 9738, which displayed strong transient activity with associated geomagnetic effects from September to December, 2001. By studying the development of spot-group and line-of-sight magnetic field together with the evolution of Hα filaments, the EUV and X-ray corona (TRACE 171 Å, Yohkoh/SXT), we have found that the magnetic structure of the active region exhibited a continuous clockwise rotation throughout its entire life. Vector magnetic data obtained from Huairou Solar Observing Station (HSOS) and full-disk line-of-sight magnetograms from SOHO/MDI allowed the determination of the best-fit force-free parameter (proxy of twist), αbest, and the systematic tilt angle (proxy of writhe) which were both found to take positive values. Soft X-ray coronal loops from Yohkoh/SXT displayed a pronounced forward-sigmoid structure in period of NOAA 9704. These observations imply that the magnetic flux tube (loops) with the same handedness (right) of the writhe and the twist rotated clockwise in the solar atmosphere for a long time. We argue that the continuous clockwise rotation of the long-lived active region may be a manifestation that a highly right-hand twisted and kinked flux tube was emerging through the photosphere and chromosphere into the corona.

  7. OBSERVATIONS OF MULTIPLE SURGES ASSOCIATED WITH MAGNETIC ACTIVITIES IN AR 10484 ON 2003 OCTOBER 25

    SciTech Connect

    Uddin, Wahab; Srivastava, Abhishek K.; Schmieder, B.; Chandra, R.; Bisht, S.; Kumar, Pankaj

    2012-06-10

    We present a multi-wavelength study of recurrent surges observed in H{alpha}, UV (Solar and Heliospheric Observatory (SOHO)/EIT), and Radio (Learmonth, Australia) from the super-active region NOAA 10484 on 2003 October 25. Several bright structures visible in H{alpha} and UV corresponding to subflares are also observed at the base of each surge. Type III bursts are triggered and RHESSI X-ray sources are evident with surge activity. The major surge consists of bunches of ejective paths forming a fan-shaped region with an angular size of ( Almost-Equal-To 65 Degree-Sign ) during its maximum phase. The ejection speed reaches up to {approx}200 km s{sup -1}. The SOHO/Michelson Doppler Imager magnetograms reveal that a large dipole emerges from the east side of the active region on 2003 October 18-20, a few days before the surges. On 2003 October 25, the major sunspots were surrounded by 'moat regions' with moving magnetic features (MMFs). Parasitic fragmented positive polarities were pushed by the ambient dispersion motion of the MMFs and annihilated with negative polarities at the borders of the moat region of the following spot to produce flares and surges. A topology analysis of the global Sun using Potential Field Source Surface shows that the fan structures visible in the EIT 171 A images follow magnetic field lines connecting the present active region to a preceding active region in the southeast. Radio observations of Type III bursts indicate that they are coincident with the surges, suggesting that magnetic reconnection is the driver mechanism. The magnetic energy released by the reconnection is transformed into plasma heating and provides the kinetic energy for the ejections. A lack of a radio signature in the high corona suggests that the surges are confined to follow the closed field lines in the fans. We conclude that these cool surges may have some local heating effects in the closed loops, but probably play a minor role in global coronal heating and the

  8. Combined experimental and modeling studies of microwave activated CH4/H2/Ar plasmas for microcrystalline, nanocrystalline, and ultrananocrystalline diamond deposition

    NASA Astrophysics Data System (ADS)

    Richley, James C.; Fox, Oliver J. L.; Ashfold, Michael N. R.; Mankelevich, Yuri A.

    2011-03-01

    A comprehensive study of microwave (MW) activated CH4/H2/Ar plasmas used for diamond chemical vapor deposition is reported, focusing particularly on the effects of gross variations in the H2/Ar ratio in the input gas mixture (from H2/Ar mole fraction ratios of > 10:1, through to ˜1:99). Absolute column densities of C2(a) and CH(X) radicals and of H(n = 2) atoms have been determined by cavity ringdown spectroscopy, as functions of height (z) above a substrate and of process conditions (CH4, H2, and Ar input mole fractions, total pressure, p, and input microwave power, P). Optical emission spectroscopy has also been used to explore the relative densities of electronically excited H atoms, and CH, C2, and C3 radicals, as functions of these same process conditions. These experimental data are complemented by extensive 2D (r, z) modeling of the plasma chemistry, which provides a quantitative rationale for all of the experimental observations. Progressive replacement of H2 by Ar (at constant p and P) leads to an expanded plasma volume. Under H2-rich conditions, > 90% of the input MW power is absorbed through rovibrational excitation of H2. Reducing the H2 content (as in an Ar-rich plasma) leads to a reduction in the absorbed power density; the plasma necessarily expands in order to accommodate a given input power. The average power density in an Ar-rich plasma is much lower than that in an H2-rich plasma operating at the same p and P. Progressive replacement of H2 by Ar is shown also to result in an increased electron temperature, an increased [H]/[H2] number density ratio, but little change in the maximum gas temperature in the plasma core (which is consistently ˜3000 K). Given the increased [H]/[H2] ratio, the fast H-shifting (CyHx + H ↔ CyHx-1 + H2; y = 1-3) reactions ensure that the core of Ar-rich plasma contains much higher relative abundances of "product" species like C atoms, and C2, and C3 radicals. The effects of Ar dilution on the absorbed power

  9. Alternative splicing interference by xenobiotics.

    PubMed

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

  10. A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication.

    PubMed

    Nordmann, Alexandra; Wixler, Ludmilla; Boergeling, Yvonne; Wixler, Viktor; Ludwig, Stephan

    2012-03-01

    Guanylate-binding proteins (GBPs) belong to the family of large GTPases that are induced in response to interferons. GBPs contain an N-terminal globular GTPase domain and a C-terminal α-helical regulatory domain that are connected by a short middle domain. Antiviral activity against vesicular stomatitis virus and encephalomyocarditis virus has been shown for hGBP-1; however, no anti-influenza virus properties for GBPs have been described to date. Here we show that hGBP-1 and hGBP-3 possess anti-influenza viral activity. Furthermore, we have identified a novel splice variant of hGBP-3, named hGBP-3ΔC, with a largely modified C-terminal α-helical domain. While all three GBP isoforms were up-regulated on influenza virus infection, hGBP-3ΔC showed the most prominent antiviral activity in epithelial cells. Mutational analysis of hGBPs revealed that the globular domain is the principal antiviral effector domain, and GTP-binding, but not hydrolysis, is necessary for antiviral action. Furthermore, we showed that hGBP-3ΔC strongly represses the activity of the viral polymerase complex, which results in decreased synthesis of viral vRNA, cRNA, mRNA, and viral proteins, as well.

  11. CALCULATING ENERGY STORAGE DUE TO TOPOLOGICAL CHANGES IN EMERGING ACTIVE REGION NOAA AR 11112

    SciTech Connect

    Tarr, Lucas; Longcope, Dana

    2012-04-10

    The minimum current corona model provides a way to estimate stored coronal energy using the number of field lines connecting regions of positive and negative photospheric flux. This information is quantified by the net flux connecting pairs of opposing regions in a connectivity matrix. Changes in the coronal magnetic field, due to processes such as magnetic reconnection, manifest themselves as changes in the connectivity matrix. However, the connectivity matrix will also change when flux sources emerge or submerge through the photosphere, as often happens in active regions. We have developed an algorithm to estimate the changes in flux due to emergence and submergence of magnetic flux sources. These estimated changes must be accounted for in order to quantify storage and release of magnetic energy in the corona. To perform this calculation over extended periods of time, we must additionally have a consistently labeled connectivity matrix over the entire observational time span. We have therefore developed an automated tracking algorithm to generate a consistent connectivity matrix as the photospheric source regions evolve over time. We have applied this method to NOAA Active Region 11112, which underwent a GOES M2.9 class flare around 19:00 on 2010 October 16th, and calculated a lower bound on the free magnetic energy buildup of {approx}8.25 Multiplication-Sign 10{sup 30} erg over 3 days.

  12. Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

    SciTech Connect

    Wadelius, C.; Lagerkvist, A. Uppsala Univ. ); Molin, A.K.; Larsson, A. ); Von Doebeln, U. )

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

  13. Starvation actively inhibits splicing of glucose-6-phosphate dehydrogenase mRNA via a bifunctional ESE/ESS element bound by hnRNP K.

    PubMed

    Cyphert, T J; Suchanek, A L; Griffith, B N; Salati, L M

    2013-09-01

    Regulated expression of glucose-6-phosphate dehydrogenase (G6PD) is due to changes in the rate of pre-mRNA splicing and not changes in its transcription. Starvation alters pre-mRNA splicing by decreasing the rate of intron removal, leading to intron retention and a decrease in the accumulation of mature mRNA. A regulatory element within exon 12 of G6PD pre-mRNA controls splicing efficiency. Starvation caused an increase in the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and this increase coincided with the increase in the binding of hnRNP K to the regulatory element and a decrease in the expression of G6PD mRNA. HnRNP K bound to two C-rich motifs forming an ESS within exon 12. Overexpression of hnRNP K decreased the splicing and expression of G6PD mRNA, while siRNA-mediated depletion of hnRNP K caused an increase in the splicing and expression of G6PD mRNA. Binding of hnRNP K to the regulatory element was enhanced in vivo by starvation coinciding with a decrease in G6PD mRNA. HnRNP K binding to the C-rich motifs blocked binding of serine-arginine rich, splicing factor 3 (SRSF3), a splicing enhancer. Thus hnRNP K is a nutrient regulated splicing factor responsible for the inhibition of the splicing of G6PD during starvation.

  14. hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells.

    PubMed

    Chou, M Y; Rooke, N; Turck, C W; Black, D L

    1999-01-01

    The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

  15. Positive control of pre-mRNA splicing in vitro.

    PubMed

    Tian, M; Maniatis, T

    1992-04-10

    Positive control of the sex-specific alternative splicing of doublesex (dsx) precursor messenger RNA (pre-mRNA) in Drosophila melanogaster involves the activation of a female-specific 3' splice site by the products of the transformer (tra) and transformer-2 (tra-2) genes. The mechanisms of this process were investigated in an in vitro system in which the female-specific 3' splice site could be activated by recombinant Tra or Tra-2 (or both). An exon sequence essential for regulation in vivo was shown to be both necessary and sufficient for activation in vitro. Nuclear proteins in addition to Tra and Tra-2 were found to bind specifically to this exon sequence. Therefore, Tra and Tra-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery.

  16. PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response.

    PubMed

    Zhang, Manchao; Deng, Youping; Riedel, Heimo

    2008-05-01

    A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.

  17. Vitamin D and alternative splicing of RNA.

    PubMed

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  18. Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: A novel epigenetic-targeting approach in acute myeloid leukemia

    PubMed Central

    Mims, Alice; Walker, Alison R.; Huang, Xiaomeng; Sun, Jin; Wang, Hongyan; Santhanam, Ramasamy; Dorrance, Adrienne M.; Walker, Chris; Hoellerbauer, Pia; Tarighat, Somayeh S.; Chan, Kenneth K.; Klisovic, Rebecca B.; Perrotti, Danilo; Caligiuri, Michael A.; Byrd, John C.; Chen, Ching-Shih; Lee, L. James; Jacob, Samson; Mrózek, Krzysztof; Bloomfield, Clara D.; Blum, William; Garzon, Ramiro; Schwind, Sebastian; Marcucci, Guido

    2013-01-01

    Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously we demonstrated that AML patients with higher miR-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (i.e., SP1, DNMT1, DNMT3A, and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represents a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDAC inhibitors. PMID:23178755

  19. Fluorinated analogues of marsanidine, a highly α2-AR/imidazoline I1 binding site-selective hypotensive agent. Synthesis and biological activities.

    PubMed

    Wasilewska, Aleksandra; Sączewski, Franciszek; Hudson, Alan L; Ferdousi, Mehnaz; Scheinin, Mika; Laurila, Jonne M; Rybczyńska, Apolonia; Boblewski, Konrad; Lehmann, Artur

    2014-11-24

    The aim of these studies was to establish the influence of fluorination of the indazole ring on the pharmacological properties of two selective α2-adrenoceptor (α2-AR) agonists: 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine, A) and its methylene analogue 1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (B). Introduction of fluorine into the indazole ring of A and B reduced both binding affinity and α2-AR/I1 imidazoline binding site selectivity. The most α2-AR-selective ligands were 6-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c) and 7-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6d). The in vivo cardiovascular properties of fluorinated derivatives of A and B revealed that in both cases the C-7 fluorination leads to compounds with the highest hypotensive and bradycardic activities. The α2-AR partial agonist 6c was prepared as a potential lead compound for development of a radiotracer for PET imaging of brain α2-ARs.

  20. Validation of the Soil Moisture Active Passive mission using USDA-ARS experimental watersheds

    NASA Astrophysics Data System (ADS)

    Cosh, M. H.; Jackson, T. J.; Bindlish, R.; Colliander, A.; Kim, S.; Das, N. N.; Yueh, S. H.; Bosch, D. D.; Goodrich, D. C.; Prueger, J. H.; Starks, P. J.; Livingston, S.; Seyfried, M. S.; Coopersmith, E. J.

    2015-12-01

    The calibration and validation program of the Soil Moisture Active Passive mission (SMAP) relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The USDA Agricultural Research Service operates several experimental watersheds which contribute to the validation of SMAP soil moisture products. These watersheds consist of a network of in situ sensors that measure soil moisture at a variety of depths including the 5 cm depth, which is critical for satellite validation. Comparisons of the in situ network estimates to the satellite products are ongoing, but initial results have shown strong correlation between satellite estimates and in situ soil moisture measurements once scaling functions were applied. The scaling methodologies for the in situ networks are being reviewed and evaluated. Results from the Little Washita, Fort Cobb, St. Joseph's and Little River Experimental Watersheds show good agreement between the satellite products and in situ measurements. Walnut Gulch results show high accuracy, although with the caveat that these domains are semi-arid with a substantially lower dynamic range. The South Fork Watershed is examined more closely for its detailed scaling function development as well as an apparent bias between satellite and in situ values.

  1. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  2. AR Signaling in Breast Cancer

    PubMed Central

    Rahim, Bilal; O’Regan, Ruth

    2017-01-01

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies. PMID:28245550

  3. AR Signaling in Breast Cancer.

    PubMed

    Rahim, Bilal; O'Regan, Ruth

    2017-02-24

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies.

  4. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    PubMed

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  5. The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

    PubMed Central

    Ziober, B L; Chen, Y; Kramer, R H

    1997-01-01

    The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair. Images PMID:9307969

  6. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

  7. Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity.

    PubMed

    Wang, Danxin; Poi, Ming J; Sun, Xiaochun; Gaedigk, Andrea; Leeder, J Steven; Sadee, Wolfgang

    2014-01-01

    Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 25% of clinically used drugs. Genetic polymorphisms cause substantial variation in CYP2D6 activity and serve as biomarkers guiding drug therapy. However, genotype-phenotype relationships remain ambiguous except for poor metabolizers carrying null alleles, suggesting the presence of yet unknown genetic variants. Searching for regulatory CYP2D6 polymorphisms, we find that a SNP defining the CYP2D6*2 allele, rs16947 [R296C, 17-60% minor allele frequency (MAF)], previously thought to convey normal activity, alters exon 6 splicing, thereby reducing CYP2D6 expression at least 2-fold. In addition, two completely linked SNPs (rs5758550/rs133333, MAF 13-42%) increase CYP2D6 transcription more than 2-fold, located in a distant downstream enhancer region (>100 kb) that interacts with the CYP2D6 promoter. In high linkage disequilibrium (LD) with each other, rs16947 and the enhancer SNPs form haplotypes that affect CYP2D6 enzyme activity in vivo. In a pediatric cohort of 164 individuals, rs16947 alone (minor haplotype frequency 28%) was associated with reduced CYP2D6 metabolic activity (measured as dextromethorphan/metabolite ratios), whereas rs5758550/rs133333 alone (frequency 3%) resulted in increased CYP2D6 activity, while haplotypes containing both rs16947 and rs5758550/rs133333 were similar to the wild-type. Other alleles used in biomarker panels carrying these variants such as CYP2D6*41 require re-evaluation of independent effects on CYP2D6 activity. The occurrence of two regulatory variants of high frequency and in high LD, residing on a long haplotype, highlights the importance of gene architecture, likely shaped by evolutionary selection pressures, in determining activity of encoded proteins.

  8. SpliceDisease database: linking RNA splicing and disease.

    PubMed

    Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua

    2012-01-01

    RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation, we give direct links of the entry to the respective position/region in the genome browser. The users can freely browse, search and download the data in SpliceDisease at http://cmbi.bjmu.edu.cn/sdisease.

  9. KIT GNNK splice variants: Expression in systemic mastocytosis and influence on the activating potential of the D816V mutation in mast cells

    PubMed Central

    Chan, Eunice Ching; Bai, Yun; Bandara, Geethani; Simakova, Olga; Brittain, Erica; Scott, Linda; Dyer, Kimberly D.; Klion, Amy D.; Maric, Irina; Gilfillan, Alasdair M.; Metcalfe, Dean D.; Wilson, Todd M.

    2013-01-01

    Stem cell factor–dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK− and GNNK+ transcripts from bone marrow mononuclear cells was developed. The GNNK−/GNNK+ copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK−/GNNK+ copy number ratio. Relative expression of only the GNNK− variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK− isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK− isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK− variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis. PMID:23743299

  10. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

    PubMed

    Listerman, Imke; Sapra, Aparna K; Neugebauer, Karla M

    2006-09-01

    Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

  11. Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825* and activating defense-related genes in Arabidopsis

    PubMed Central

    Niu, Dongdong; Xia, Jing; Jiang, Chunhao; Qi, Beibei; Ling, Xiaoyu; Lin, Siyuan; Zhang, Weixiong; Guo, Jianhua; Jin, Hailing; Zhao, Hongwei

    2016-01-01

    Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment.miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted. PMID:26526683

  12. Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825* and activating defense-related genes in Arabidopsis.

    PubMed

    Niu, Dongdong; Xia, Jing; Jiang, Chunhao; Qi, Beibei; Ling, Xiaoyu; Lin, Siyuan; Zhang, Weixiong; Guo, Jianhua; Jin, Hailing; Zhao, Hongwei

    2016-04-01

    Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment. miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted.

  13. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  14. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGES

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; ...

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  15. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  16. Discovery of 34 g ,mCl (p,γ ) 35Ar resonances activated at classical nova temperatures

    NASA Astrophysics Data System (ADS)

    Fry, C.; Wrede, C.; Bishop, S.; Brown, B. A.; Chen, A. A.; Faestermann, T.; Hertenberger, R.; Parikh, A.; Pérez-Loureiro, D.; Wirth, H.-F.; García, A.; Ortez, R.

    2015-01-01

    Background: The thermonuclear 34 g ,mCl (p,γ ) 35Ar reaction rates are unknown due to a lack of experimental nuclear physics data. Uncertainties in these rates translate to uncertainties in 34S production in models of classical novae on oxygen-neon white dwarfs. 34S abundances have the potential to aid in the classification of presolar grains. Purpose: Determine resonance energies for the 34 g ,mCl (p,γ ) 35Ar reactions within the region of astrophysical interest for classical novae to a precision of a few keV as an essential first step toward constraining their thermonuclear reaction rates. Method: 35Ar excited states were populated by the 36Ar (d,t ) 35Ar reaction at E (d )=22 MeV and reaction products were momentum analyzed by a high resolution quadrupole-dipole-dipole-dipole (Q3D) magnetic spectrograph. Results: Seventeen new 35Ar levels have been detected at a statistically significant level in the region Ex≈5.9 -6.7 MeV (Er<800 keV ) and their excitation energies have been determined to typical uncertainties of 3 keV. The uncertainties for five previously known levels have also been reduced substantially. The measured level density was compared to those calculated using the WBMB Hamiltonian within the s d -p f model space. Conclusions: Most of the resonances in the region of astrophysical interest have likely been discovered and their energies have been determined, but the resonance strengths are still unknown, and experimentally constraining the 34 g ,mCl (p,γ ) 35Ar reaction rates will require further experiments.

  17. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    PubMed Central

    Balestra, Dario; Scalet, Daniela; Pagani, Franco; Rogalska, Malgorzata Ewa; Mari, Rosella; Bernardi, Francesco; Pinotti, Mirko

    2016-01-01

    In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients. PMID:27701399

  18. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*

    PubMed Central

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

    2015-01-01

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  19. Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma

    PubMed Central

    2013-01-01

    Background Multiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells. Findings We have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells. Conclusions This is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease. PMID:24280290

  20. Functional impact of splice isoform diversity in individual cells

    PubMed Central

    Yap, Karen; Makeyev, Eugene V.

    2016-01-01

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

  1. Functional impact of splice isoform diversity in individual cells.

    PubMed

    Yap, Karen; Makeyev, Eugene V

    2016-08-15

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities.

  2. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  3. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  4. Alternative Splicing of STAT3 Is Affected by RNA Editing.

    PubMed

    Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

    2017-03-09

    A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

  5. Ar-Ar_Redux: rigorous error propagation of 40Ar/39Ar data, including covariances

    NASA Astrophysics Data System (ADS)

    Vermeesch, P.

    2015-12-01

    Rigorous data reduction and error propagation algorithms are needed to realise Earthtime's objective to improve the interlaboratory accuracy of 40Ar/39Ar dating to better than 1% and thereby facilitate the comparison and combination of the K-Ar and U-Pb chronometers. Ar-Ar_Redux is a new data reduction protocol and software program for 40Ar/39Ar geochronology which takes into account two previously underappreciated aspects of the method: 1. 40Ar/39Ar measurements are compositional dataIn its simplest form, the 40Ar/39Ar age equation can be written as: t = log(1+J [40Ar/39Ar-298.5636Ar/39Ar])/λ = log(1 + JR)/λ Where λ is the 40K decay constant and J is the irradiation parameter. The age t does not depend on the absolute abundances of the three argon isotopes but only on their relative ratios. Thus, the 36Ar, 39Ar and 40Ar abundances can be normalised to unity and plotted on a ternary diagram or 'simplex'. Argon isotopic data are therefore subject to the peculiar mathematics of 'compositional data', sensu Aitchison (1986, The Statistical Analysis of Compositional Data, Chapman & Hall). 2. Correlated errors are pervasive throughout the 40Ar/39Ar methodCurrent data reduction protocols for 40Ar/39Ar geochronology propagate the age uncertainty as follows: σ2(t) = [J2 σ2(R) + R2 σ2(J)] / [λ2 (1 + R J)], which implies zero covariance between R and J. In reality, however, significant error correlations are found in every step of the 40Ar/39Ar data acquisition and processing, in both single and multi collector instruments, during blank, interference and decay corrections, age calculation etc. Ar-Ar_Redux revisits every aspect of the 40Ar/39Ar method by casting the raw mass spectrometer data into a contingency table of logratios, which automatically keeps track of all covariances in a compositional context. Application of the method to real data reveals strong correlations (r2 of up to 0.9) between age measurements within a single irradiation batch. Propertly taking

  6. Spectroscopic and modeling investigations of the gas-phase chemistry and composition in microwave plasma activated B2H6/Ar/H2 mixtures.

    PubMed

    Ma, Jie; Richley, James C; Davies, David R W; Cheesman, Andrew; Ashfold, Michael N R; Mankelevich, Yuri A

    2010-02-25

    This paper describes a three-pronged study of microwave (MW) activated B(2)H(6)/Ar/H(2) plasmas as a precursor to diagnosis of the B(2)H(6)/CH(4)/Ar/H(2) plasmas used for the chemical vapor deposition of B-doped diamond. Absolute column densities of B atoms and BH radicals have been determined by cavity ring-down spectroscopy as a function of height (z) above a molybdenum substrate and of the plasma process conditions (B(2)H(6) and Ar partial pressures, total pressure, and supplied MW power). Optical emission spectroscopy has been used to explore variations in the relative densities of electronically excited BH, H, and H(2) species as a function of the same process conditions and of time after introducing B(2)H(6) into a pre-existing Ar/H(2) plasma. The experimental measurements are complemented by extensive 2-D(r, z) modeling of the plasma chemistry, which results in refinements to the existing B/H chemistry and thermochemistry and demonstrates the potentially substantial loss of gas-phase BH(x) species through reaction with trace quantities of air/O(2) in the process gas mixture and heterogeneous processes occurring at the reactor wall.

  7. Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of Helicobacter pylori

    PubMed Central

    Pflock, Michael; Kennard, Simone; Delany, Isabel; Scarlato, Vincenzo; Beier, Dagmar

    2005-01-01

    The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein. PMID:16177315

  8. Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

    PubMed

    Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2016-02-01

    We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals.

  9. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  10. Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression.

    PubMed

    Wang, Xueting; Pan, Longlu; Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

    2017-03-07

    Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis.

  11. Transcription and splicing: when the twain meet.

    PubMed

    Brody, Yehuda; Shav-Tal, Yaron

    2011-01-01

    Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

  12. Activation of thermosensitive RNA splicing and production of a heat-labile P85gag-mos kinase by the introduction of a specific deletion in murine sarcoma virus-124 DNA.

    PubMed Central

    de Mars, M; Cizdziel, P E; Murphy, E C

    1988-01-01

    Murine sarcoma virus ts110 (MuSVts110) is a conditionally transformation-defective MuSV mutant lacking 1,487 bases found in its wild-type parent, MuSV-349 (MuSV-124). Expression of the MuSVts110 v-mos gene product, P85gag-mos, requires splicing of the viral transcript to align the gag and mos genes in frame. However, this splice event is restricted to growth temperatures of 33 degrees C or lower. No splicing of the viral RNA, no production of P85gag-mos, and, hence, no cell transformation is observed at growth temperatures above 33 degrees C. To determine whether thermosensitive splicing is an intrinsic property of To determine whether thermosensitive splicing is an intrinsic property of MuSVts110 RNA specified by the 1,487-base deletion or a result of a cellular defect, we examined an "equivalent" or MuSVts110 DNA (designated ts32 DNA) constructed by combining wild-type MuSV-124 DNA fragments with a synthetic oligonucleotide to yield an otherwise wild-type viral DNA containing the same 1,487-base deletion as authentic MuSVts110. As observed in control cells (6m2 cells) infected with the authentic MuSVts110 virus, NIH 3T3 cells transfected with ts32 DNA appeared morphologically transformed when grown at 33 degrees C, but were converted to a more normal, flattened shape within a few hours of a shift to 39 degrees C. In concert with these morphological changes, both the processing of the ts32 RNA transcripts and the production of ts32 p85gag-mos kinase were found to be optimal at growth temperatures from 28 to 33 degrees C, but dramatically reduced at 37 to 41 degrees C. Like authentic P85gag-mos, the ts32 P85gag-mos kinase activity was rapidly inactivated by brief exposure to 39 degrees C. These results suggested that the MuSVts110 equivalent is functionally indistinguishable from authentic MuSVts110 and that the novel temperature-sensitive splicing of MuSVts110 transcripts is specified by an intrinsic property of the viral RNA. Images PMID:2835496

  13. Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity.

    PubMed

    Macdonald, Patrick J; Francy, Christopher A; Stepanyants, Natalia; Lehman, Lance; Baglio, Anthony; Mears, Jason A; Qi, Xin; Ramachandran, Rajesh

    2016-01-01

    Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.

  14. Paleomagnetic and 40Ar/39Ar results from the Grant intrusive breccia and coparison to the Permian Downeys Bluff Sill; evidence for Permian igneous activity at Hicks Dome, southern Illinois Basin

    USGS Publications Warehouse

    Reynolds, Richard L.; Goldhaber, Martin B.; Snee, Lawrence W.

    1997-01-01

    Igneous processes at Hicks dome, a structural upwarp at lat 37.5 degrees N., long 88.4 degrees W. in the southern part of the Illinois Basin, may have thermally affected regional basinal fluid flow and may have provided fluorine for the formation of the Illinois-Kentucky Fluorspar district. The timing of both igneous activity and mineralization is poorly known. For this reason, we have dated an intrusive breccia at Hicks dome, the Grant intrusion, using 40Ar/39Ar geochronometric and paleomagnetic methods. Concordant plateau dates, giving Permian ages, were obtained from amphibole (272.1+or-0.7 [1 sigma] Ma) and phlogopite (272.7+or-0.7 [1 sigma] Ma). After alternating-field (AF) demagnetization, specimens that contain titanomagnetite-bearing igneous rock fragments give a mean remanent direction of declination (D)=168.4 degrees; inclination (I)=-8 degrees; alpha 95=8.6 degrees; number of specimens (N)=10; this direction yields a virtual geomagnetic pole (VGP) at lat 54.8 degrees N., long 119.0 degrees E., delta p=4.4 degrees, delta m=8.7 degrees, near the late Paleozoic part of the North American apparent pole wander path. A nearly identical magnetization was found for the nearby Downeys Bluff sill (previously dated at about 275+or-24 Ma by the Rb-Sr method), in southern Illinois. Both AF and thermal demagnetization isolated shallow, southeasterly remanent directions carried by magnetite in the sill and from pyrrhotite in the baked contact of the Upper Mississippian Downeys Bluff Limestone: D=158.6 degrees; I=-11.8 degrees; alpha 95=3.8 degrees; N=15, yielding a VGP at lat 53.0 degrees N., long 128.7 degrees E., delta p=2.0 degrees, delta m=3.9 degrees. The paleomagnetic results, isotopic dates, and petrographic evidence thus favor the acquisition of thermal remanent magnetization by the Grant breccia and the Downeys Bluff sill during the Permian. The isotopic dates record rapid cooling from temperatures greater than 550 degrees C to less than 300 degrees C (the

  15. RNA splicing: disease and therapy.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2011-05-01

    The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.

  16. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  17. A genome-wide siRNA screen for regulators of tumor suppressor p53 activity in human non-small lung cancer cells identifies components of the RNA splicing machinery as targets for anticancer treatment.

    PubMed

    Siebring-van Olst, Ellen; Blijlevens, Maxime; de Menezes, Renee X; van der Meulen-Muileman, Ida H; Smit, Egbert F; van Beusechem, Victor W

    2017-03-13

    Reinstating wild-type tumor suppressor p53 activity could be a valuable option for the treatment of cancer. To contribute to development of new treatment options for non-small cell lung cancer (NSCLC), we performed genome-wide siRNA screens for determinants of p53 activity in NSCLC cells. We identified many genes not previously known to be involved in regulating p53 activity. Silencing p53 pathway inhibitor genes was associated with loss of cell viability. The largest functional gene cluster influencing p53 activity was mRNA splicing. Prominent p53 activation was observed upon silencing of specific spliceosome components, rather than by general inhibition of the spliceosome. Ten genes were validated as inhibitors of p53 activity in multiple NSCLC cell lines: genes encoding the Ras-pathway activator SOS1, the zinc finger protein TSHZ3, the mitochondrial membrane protein COX16 and the spliceosome components SNRPD3, SF3A3, SF3B1, SF3B6, XAB2, CWC22 and HNRNPL. Silencing these genes generally increased p53 levels, with distinct effects on CDKN1A expression, induction of cell cycle arrest and cell death. Silencing spliceosome components was associated with alternative splicing of MDM4 mRNA, which could contribute to activation of p53. In addition, silencing splice factors was particularly effective in killing NSCLC cells, albeit in a p53-independent manner. Interestingly, silencing SNRPD3 and SF3A3 exerted much stronger cytotoxicity to NSCLC cells than to lung fibroblasts, suggesting that these genes could represent useful therapeutic targets. This article is protected by copyright. All rights reserved.

  18. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  19. Alternative RNA splicing and cancer.

    PubMed

    Liu, Sali; Cheng, Chonghui

    2013-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells.

  20. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    PubMed

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues.

  1. Activation of a cryptic splice site in the growth hormone receptor associated with growth hormone insensitivity syndrome in a genetic isolate of Laron Syndrome

    SciTech Connect

    Schiavi, A.; Bartlett, R.; Brown, M.

    1994-09-01

    Laron syndrome (LS) is a rare, autosomal recessive disease found worldwide. Despite various ethnic differences, all patients with LS described display classic dysmorphic features and extreme short stature due to defects in the growth hormone receptor (GHR). The vast majority of these patients are sporadic occurrences resulting from consanguineous matings; however, an Ecuadorian genetic isolate of LS has been reported. Our investigations have identified a genetic isolate of LS of Anglo Saxon origin. Seven individuals, by all clinical and biochemical criteria, have LS. As a result of extensive review of family and medical histories we have constructed a pedigree tracing the lineage of our affected patients through the 17th century. No GHR gross deletions were detected using an exon-specific PCR assay developed in our laboratory. Previous molecular analyses have identified mutations in exons 2-7 in numerous patients with classical LS. Single strand conformational polymorphism (SSCP) analysis was performed on GHR exons 2-7, and a marked conformational shift was noted in exon 7. Cycle sequencing of exon 7 from three affected individuals, and from four first-degree relatives, revealed a C{r_arrow}T transition at position 766 of the cDNA, and a heterozygous C{r_arrow}T transition at the identical position in the obligate carriers studied. This mutation is predicted to activate a cryptic donor splice site 63 base pairs upstream from the 3{prime} end of exon 7, effectively truncating the GHR cDNA without changing the reading frame. The resultant GHR protein is shortened by a proposed 21 amino acids. The identification and conformation of this mutation not only identifies a novel mutation in the GHR, and the first to be described in LS patients of English descent, but also allows for comparisons between genotypes and phenotypes in an inbred population.

  2. The Intronic GABRG2 Mutation, IVS6+2T→G, Associated with CAE Altered Subunit mRNA Intron Splicing, Activated Nonsense-Mediated Decay and Produced a Stable Truncated γ2 Subunit

    PubMed Central

    Tian, Mengnan; Macdonald, Robert L.

    2012-01-01

    The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy (CAE) and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wildtype and mutant γ2S subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wildtype and mutant γ2S subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wildtype and mutant GABRG2 mRNA splicing patterns were determined in both BAC transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention and produced a frame shift in exon 7 that created a premature translation-termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense mediated mRNA decay (NMD) or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first 6 GABRG2 exons and a novel frame-shifted 29 aa C terminal tail. The γ2-PTC subunit was homologous to the mollusk acetylcholine binding protein (AChBP) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant negative effect on αβγ2 receptor assembly. PMID:22539854

  3. Independent CMEs from a Single Solar Active Region - The Case of the Super-Eruptive NOAA AR11429

    NASA Astrophysics Data System (ADS)

    Chintzoglou, Georgios; Patsourakos, Spiros; Vourlidas, Angelos

    2014-06-01

    In this investigation we study AR 11429, the origin of the twin super-fast CME eruptions of 07-Mar-2012. This AR fulfills all the requirements for the 'perfect storm'; namely, Hale's law incompatibility and a delta-magnetic configuration. In fact, during its limb-to-limb transit, AR 11429 spawned several eruptions which caused geomagnetic storms, including the biggest in Cycle 24 so far. Magnetic Flux Ropes (MFRs) are twisted magnetic structures in the corona, best seen in ~10MK hot plasma emission and are often considered as the culprit causing such super-eruptions. However, their 'dormant' existence in the solar atmosphere (i.e. prior to eruptions), is a matter of strong debate. Aided by multi-wavelength and multi-spacecraft observations (SDO/HMI & AIA, HINODE/SOT/SP, STEREO B/EUVI) and by using a Non-Linear Force-Free (NLFFF) model for the coronal magnetic field, our work shows two separate, weakly-twisted magnetic flux systems which suggest the existence of possible pre-eruption MFRs.

  4. Synergistic Effect of Simple Sugars and Carboxymethyl Cellulose on the Production of a Cellulolytic Cocktail from Bacillus sp. AR03 and Enzyme Activity Characterization.

    PubMed

    Manfredi, Adriana P; Pisa, José H; Valdeón, Daniel H; Perotti, Nora I; Martínez, María A

    2016-04-01

    A cellulase-producing bacterium isolated from pulp and paper feedstock, Bacillus sp. AR03, was evaluated by means of a factorial design showing that peptone and carbohydrates were the main variables affecting enzyme production. Simple sugars, individually and combined with carboxymethyl cellulose (CMC), were further examined for their influence on cellulase production by strain AR03. Most of the mono and disaccharides assayed presented a synergistic effect with CMC. As a result, a peptone-based broth supplemented with 10 g/L sucrose and 10 g/L CMC maximized enzyme production after 96 h of cultivation. This medium was used to produce endoglucanases in a 1-L stirred tank reactor in batch mode at 30 °C, which reduced the fermentation period to 48 h and reaching 3.12 ± 0.02 IU/mL of enzyme activity. Bacillus sp. AR03 endoglucanases showed an optimum temperature of 60 °C and a pH of 6.0 with a wide range of pH stability. Furthermore, presence of 10 mM Mn(2+) and 5 mM Co(2+) produced an increase of enzyme activity (246.7 and 183.7 %, respectively), and remarkable tolerance to NaCl, Tween 80, and EDTA was also observed. According to our results, the properties of the cellulolytic cocktail from Bacillus sp. AR03 offer promising features in view of potential biorefinery applications.

  5. Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells.

    PubMed

    Dietze, Raimund; Konrad, Lutz; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-03-01

    Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.

  6. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

  7. Tau mis-splicing in the pathogenesis of neurodegenerative disorders

    PubMed Central

    Park, Sun Ah; Ahn, Sang Il; Gallo, Jean-Marc

    2016-01-01

    Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413] PMID:27222125

  8. Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

    PubMed Central

    Turton, Keren B.; Esnault, Stephane; Delain, Larissa P.; Mosher, Deane F.

    2016-01-01

    Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites. PMID:27768061

  9. Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing.

    PubMed

    Kong, Ka-Yiu Edwin; Tang, Hei-Man Vincent; Pan, Kewu; Huang, Zhe; Lee, Tsz-Hang Jimmy; Hinnebusch, Alan G; Jin, Dong-Yan; Wong, Chi-Ming

    2014-01-01

    Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.

  10. Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).

    PubMed

    Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

    2013-05-01

    Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

  11. Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

    PubMed Central

    Cote, Gilbert J.; Zhu, Wen; Thomas, Anthony; Martin, Emil; Murad, Ferid; Sharina, Iraida G.

    2012-01-01

    Background Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER). Methodology/Principal Findings Here we report that splicing of C-α1 sGC can be modulated by H2O2 treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H2O2 treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H2O2 occurs at the protein level with variable regulation observed in different breast cancer cells. Conclusions/Significance Our data demonstrate that H2O2 regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H2O2 treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress. PMID:22911749

  12. Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis

    PubMed Central

    Nie, Pingping; Li, Xia; Wang, Shune; Guo, Jianhua; Zhao, Hongwei; Niu, Dongdong

    2017-01-01

    Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against Botrytis cinerea remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and B. cinerea inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in sid2-2 and NahG mutants, but not in jar1, ein2 and npr1 mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components. PMID:28293243

  13. Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis.

    PubMed

    Nie, Pingping; Li, Xia; Wang, Shune; Guo, Jianhua; Zhao, Hongwei; Niu, Dongdong

    2017-01-01

    Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against Botrytis cinerea remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and B. cinerea inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in sid2-2 and NahG mutants, but not in jar1, ein2 and npr1 mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components.

  14. Splicing of aged fibers

    NASA Astrophysics Data System (ADS)

    Volotinen, Tarja T.; Yuce, Hakan H.; Bonanno, Nicholas; Frantz, Rolf A.; Duffy, Sean

    1993-11-01

    The deployment of fiber in the subscriber loop will require that an optical fiber network maintain the highest possible level of reliability over time, despite being subjected to extremes of temperature, humidity, and other environmental and mechanical stresses imposed on the outside plant. At the same time, both the initial cost and the ongoing maintenance expenses for loop equipment must be kept low. Fiber in the Loop (FITL) applications will entail increased fiber handling. Cable lengths will be shorter, and fiber counts higher, than has been the case so far in long-distance applications. There will also be more cable sheath openings per unit length of cable and/or fiber, as well as more splicing and connectorization. It may become a common practice that a customer is connected to a cable installed many years earlier. In subscriber loops, cables and fibers will be installed in harsher and more varying environments. Fibers will be exposed to higher humidity and temperature, particularly in splice boxes mounted on building walls, in pedestal cabinets, and in other similar enclosures. Corrosive gases and/or liquids may also be present at some locations and will adversely affect the fibers. The combination of increased handling, greater exposure, and more stressful environments may give rise to a need for new, more stringent requirements for fiber mechanical reliability. These can include increaSed fiber strength, increased aging resistance, and increased fatigue resistance.

  15. ArArCALC—software for 40Ar/ 39Ar age calculations

    NASA Astrophysics Data System (ADS)

    Koppers, Anthony A. P.

    2002-06-01

    ArArCALC is a Microsoft Excel ® 97-2000-XP application for performing calculations in 40Ar/ 39Ar geochronology. It is coded in Visual Basic for Applications and can be used under the Windows ® 95/98/NT/2000/ME/XP operating systems. ArArCALC provides an easy-to-use graphical interface for the calculation of age plateaus, total fusion ages and isochrons following the regression of 40Ar/ 39Ar mass spectrometry data. Results are stored in single Excel workbooks including nine different data tables and four different diagrams. Analytical, internal and external errors are calculated based on error propagation of all input parameters, analytical data and applied corrections. Finally, the age calculation results can be recalibrated with reference to the primary K-Ar standards (e.g. GA-1550, MMhb-1) in order to obtain more consistent absolute40Ar/ 39Ar age determinations. ArArCALC is distributed as freeware.

  16. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

    PubMed Central

    Schneider, Cornelius; Agafonov, Dmitry E.; Schmitzová, Jana; Hartmuth, Klaus; Fabrizio, Patrizia; Lührmann, Reinhard

    2015-01-01

    Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast Bact spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the Bact crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing. PMID:26393790

  17. Determining K/Ar age of fault activity through analysis of clay mineralogy: A case study of "El Doctor Fault", México

    NASA Astrophysics Data System (ADS)

    Garduño, D. E.; Pi, T.; Sole, J.; Martini, M.; Alcala, J. R.

    2013-05-01

    The upper continental crust of Mexico is cut by several major faults, some of which were interpreted as terrane boundaries. Although the age of such faults is key to reconstructing the tectonic evolution of Mexico, geochronologic studies focused on the absolute dating of a fault are scattered. The Doctor fault zone is a decakilometric NNW-SSE structure that produced the overriding of the Lower Cretaceus El Doctor carbonate platform onto foreland calcareous turbidites of Upper Cretaceous Soyatal Formation. In the fault zone, turbidites of the Soyatal Formation display a pervasive foliation at the submillimeter-scale. In calcareous layers, this foliation is defined by seams of opaque minerals concentrated along stilolitic surfaces, whereas in lutitic layers it is defined by iso-oriented fine-grained illite. We collected 17 samples from a traverse across the Doctor fault zone, in order to (1) defining and quantifying fault-related changes in clay mineralogy, (2) studying fabrics in clay-rich fault rocks and protolith, and (3) dating the fault activity by illite K/Ar with laser. Texture was studied with petrographic microscope on polished thin sections. Three size fractions (from 2 μm to 0.05 μm) were extracted using centrifugation. Clay mineralogy was determined using XRD in clay oriented samples and the illite crystallinity (IC) has been determined by the Kübler method (Kisch, 1990). The amount of 2M1 illite was quantified using XRD patterns from a randomly oriented sample, achieved using WILDFIRE (Reynolds, 1994, Haines and Van der Pluijm, 2008) and RIETVELD methods and the timing of fault main activity is determined using K/Ar dating. The mineralogy of the samples consists of quartz, calcite, plagioclase, hematite and clays. The clay mineralogy contain illite (zone 1, zone 2 and zone 3), smectite (zone 2), chlorite (zone 3), kaolinite (zone 1 and zone3), and vermiculite (zone 3). The range of IC (0.24 to 0.4) is attributed to heterogeneous origins of illite

  18. Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma.

    PubMed

    Fu, Yao; Zhao, Xingli; Li, Zhaohui; Wei, Jun; Tian, Yu

    2016-08-01

    The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma.

  19. Biochemistry and regulation of pre-mRNA splicing.

    PubMed

    Adams, M D; Rudner, D Z; Rio, D C

    1996-06-01

    During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

  20. 39Ar/Ar measurements using ultra-low background proportional counters

    SciTech Connect

    Hall, Jeter C.; Aalseth, Craig E.; Bonicalzi, Ricco; Brandenberger, Jill M.; Day, Anthony R.; Humble, Paul H.; Mace, Emily K.; Panisko, Mark E.; Seifert, Allen

    2016-01-08

    Age dating groundwater and seawater using 39Ar/Ar ratios is an important tool to understand water mass flow rates and mean residence time. For modern or contemporary argon, the 39Ar activity is 1.8 mBq per liter of argon. Radiation measurements at these activity levels require ultra low-background detectors. Low-background proportional counters have been developed at Pacific Northwest National Laboratory. These detectors use traditional mixtures of argon and methane as counting gas, and the residual 39Ar from commercial argon has become a predominant source of background activity in these detectors. We demonstrated sensitivity to 39Ar by using geological or ancient argon from gas wells in place of commercial argon. The low level counting performance of these proportional counters is then demonstrated for sensitivities to 39Ar/Ar ratios sufficient to date water masses as old as 1000 years.

  1. The ArsD As(III) metallochaperone

    PubMed Central

    Ajees, A. Abdul; Yang, Jianbo

    2013-01-01

    Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four β-strands flanked by four α-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of α1 helix and metalloid binding site of ArsD. PMID:21188475

  2. Early stellate cell activation and veno-occlusive-disease (VOD)-like hepatotoxicity in dogs treated with AR-H047108, an imidazopyridine proton pump inhibitor.

    PubMed

    Berg, Anna-Lena; Böttcher, Gerhard; Andersson, Kjell; Carlsson, Enar; Lindström, Anna-Karin; Huby, Russell; Håkansson, Helen; Skånberg-Wilhelmsson, Inger; Hellmold, Heike

    2008-07-01

    Dogs treated with AR-H047108, an imidazopyridine potassium competitive acid blocker (P-CAB), developed clinical signs of hepatic dysfunction as well as morphologically manifest hepatotoxicity in repeat-dose toxicity studies. An investigative one-month study was performed, with interim euthanasia after one and two weeks. A detailed histopathological and immunohistochemical characterization of the liver lesions was conducted, including markers for fibrosis, Kupffer cell activation, apoptosis, and endothelial injury. In addition, hepatic retinoid and procollagen 1alpha2 mRNA levels in livers of dogs treated with AR-H047108 were analyzed. The results showed an early inflammatory process in central veins and centrilobular areas, present after one week of treatment. This inflammatory reaction was paralleled by activation of stellate/Ito cells to myofibroblasts and was associated with sinusoidal and centrivenular fibrosis. The early activation of stellate cells coincided with a significant decrease in retinyl ester levels, and a significant increase in procollagen 1alpha2 mRNA levels, in the liver. At later time points (three and six months), there was marked sinusoidal fibrosis in centrilobular areas, as well as occlusion of central veins resulting from a combination of fibrosis and increased thickness of smooth muscle bundles in the vessel wall. The pattern of lesions suggests a veno-occlusive-disease (VOD)-like scenario, possibly linked to the imidazopyridine chemical structure of the compound facilitated by specific morphological features of the dog liver.

  3. Functional studies on the ATM intronic splicing processing element.

    PubMed

    Lewandowska, Marzena A; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

  4. Functional studies on the ATM intronic splicing processing element

    PubMed Central

    Lewandowska, Marzena A.; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E.; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing. PMID:16030351

  5. Entropic contributions to the splicing process

    NASA Astrophysics Data System (ADS)

    Osella, Matteo; Caselle, Michele

    2009-12-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model.

  6. New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia

    PubMed Central

    Panuzzo, Cristina; Volpe, Gisella; Rocchietti, Elisa Cibrario; Casnici, Claudia; Crotta, Katia; Crivellaro, Sabrina; Carrà, Giovanna; Lorenzatti, Roberta; Peracino, Barbara; Torti, Davide; Morotti, Alessandro; Camacho-Leal, Maria Pilar; Defilippi, Paola; Marelli, Ornella; Saglio, Giuseppe

    2015-01-01

    In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies. PMID:26682280

  7. Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies.

    PubMed

    Xu, Dong-Qing; Mattox, William

    2006-01-15

    Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.

  8. The transcription factor c-Myb affects pre-mRNA splicing

    SciTech Connect

    Orvain, Christophe; Matre, Vilborg; Gabrielsen, Odd S.

    2008-07-25

    c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF{sup 65} and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.

  9. Estimation of the minimum mRNA splicing error rate in vertebrates.

    PubMed

    Skandalis, A

    2016-01-01

    The majority of protein coding genes in vertebrates contain several introns that are removed by the mRNA splicing machinery. Errors during splicing can generate aberrant transcripts and degrade the transmission of genetic information thus contributing to genomic instability and disease. However, estimating the error rate of constitutive splicing is complicated by the process of alternative splicing which can generate multiple alternative transcripts per locus and is particularly active in humans. In order to estimate the error frequency of constitutive mRNA splicing and avoid bias by alternative splicing we have characterized the frequency of splice variants at three loci, HPRT, POLB, and TRPV1 in multiple tissues of six vertebrate species. Our analysis revealed that the frequency of splice variants varied widely among loci, tissues, and species. However, the lowest observed frequency is quite constant among loci and approximately 0.1% aberrant transcripts per intron. Arguably this reflects the "irreducible" error rate of splicing, which consists primarily of the combination of replication errors by RNA polymerase II in splice consensus sequences and spliceosome errors in correctly pairing exons.

  10. The Role of Canonical and Noncanonical Pre-mRNA Splicing in Plant Stress Responses

    PubMed Central

    Dubrovina, A. S.; Kiselev, K. V.; Zhuravlev, Yu. N.

    2013-01-01

    Plants are sessile organisms capable of adapting to various environmental constraints, such as high or low temperatures, drought, soil salinity, or pathogen attack. To survive the unfavorable conditions, plants actively employ pre-mRNA splicing as a mechanism to regulate expression of stress-responsive genes and reprogram intracellular regulatory networks. There is a growing evidence that various stresses strongly affect the frequency and diversity of alternative splicing events in the stress-responsive genes and lead to an increased accumulation of mRNAs containing premature stop codons, which in turn have an impact on plant stress response. A number of studies revealed that some mRNAs involved in plant stress response are spliced counter to the traditional conception of alternative splicing. Such noncanonical mRNA splicing events include trans-splicing, intraexonic deletions, or variations affecting multiple exons and often require short direct repeats to occur. The noncanonical alternative splicing, along with common splicing events, targets the spliced transcripts to degradation through nonsense-mediated mRNA decay or leads to translation of truncated proteins. Investigation of the diversity, biological consequences, and mechanisms of the canonical and noncanonical alternative splicing events will help one to identify those transcripts which are promising for using in genetic engineering and selection of stress-tolerant plants. PMID:23509698

  11. Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes.

    PubMed Central

    Hall, C; Sin, W C; Teo, M; Michael, G J; Smith, P; Dong, J M; Lim, H H; Manser, E; Spurr, N K; Jones, T A

    1993-01-01

    n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing. Images PMID:8336731

  12. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after approximately 3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After approximately 3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of approximately 118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control

  13. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  14. Modulators of alternative splicing as novel therapeutics in cancer.

    PubMed

    Oltean, Sebastian

    2015-10-10

    Alternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g., pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics.

  15. Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing.

    PubMed

    Hovhannisyan, Ruben H; Warzecha, Claude C; Carstens, Russ P

    2006-01-01

    Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons. Such analysis included use of a novel fluorescence-based splicing reporter assay that allowed quantitative determination of relative functional activity of ISE/ISS-3 mutants using flow cytometric analysis of live cells. We determined that specific sequences within this element that mediate splicing enhancement also mediate splicing repression, depending on their position relative to a regulated exon. Thus, factors that bind the element are likely to be coordinately involved in mediating both aspects of splicing regulation. Exon IIIc silencing is dependent upon a suboptimal branchpoint sequence containing a guanine branchpoint nucleotide. Previous studies of exon IIIc splicing in HeLa nuclear extracts demonstrated that this guanine branchsite primarily impaired the second step of splicing suggesting that ISE/ISS-3 may block exon IIIc inclusion at this step. However, results presented here that include use of newly developed in vitro splicing assays of FGFR2 using extracts from a cell line expressing FGFR2-IIIb strongly suggest that cell-type-specific silencing of exon IIIc occurs at or prior to the first step of splicing.

  16. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

    PubMed

    Selvanathan, Saravana P; Graham, Garrett T; Erkizan, Hayriye V; Dirksen, Uta; Natarajan, Thanemozhi G; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T; Ljungman, Mats E; Wu, Cathy H; Lawlor, Elizabeth R; Üren, Aykut; Toretsky, Jeffrey A

    2015-03-17

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

  17. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner.

    PubMed Central

    Apt, D; Liu, Y; Bernard, H U

    1994-01-01

    Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers. Images PMID:7937100

  18. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators

    PubMed Central

    Llorian, Miriam; Gooding, Clare; Bellora, Nicolas; Hallegger, Martina; Buckroyd, Adrian; Wang, Xiao; Rajgor, Dipen; Kayikci, Melis; Feltham, Jack; Ule, Jernej; Eyras, Eduardo; Smith, Christopher W.J.

    2016-01-01

    Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery. PMID:27317697

  19. Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

    PubMed

    Papasaikas, Panagiotis; Tejedor, J Ramón; Vigevani, Luisa; Valcárcel, Juan

    2015-01-08

    Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

  20. ACTH Action on StAR Biology.

    PubMed

    Clark, Barbara J

    2016-01-01

    Adrenocorticotropin hormone (ACTH) produced by the anterior pituitary stimulates glucocorticoid synthesis by the adrenal cortex. The first step in glucocorticoid synthesis is the delivery of cholesterol to the mitochondrial matrix where the first enzymatic reaction in the steroid hormone biosynthetic pathway occurs. A key response of adrenal cells to ACTH is activation of the cAMP-protein kinase A (PKA) signaling pathway. PKA activation results in an acute increase in expression and function of the Steroidogenic Acute Regulatory protein (StAR). StAR plays an essential role in steroidogenesis- it controls the hormone-dependent movement of cholesterol across the mitochondrial membranes. Currently StAR's mechanism of action remains a major unanswered question in the field. However, some insight may be gained from understanding the mechanism(s) controlling the PKA-dependent phosphorylation of StAR at S194/195 (mouse/human StAR), a modification that is required for function. This mini-review provides a background on StAR's biology with a focus on StAR phosphorylation. The model for StAR translation and phosphorylation at the outer mitochondrial membrane, the location for StAR function, is presented to highlight a unifying theme emerging from diverse studies.

  1. ACTH Action on StAR Biology

    PubMed Central

    Clark, Barbara J.

    2016-01-01

    Adrenocorticotropin hormone (ACTH) produced by the anterior pituitary stimulates glucocorticoid synthesis by the adrenal cortex. The first step in glucocorticoid synthesis is the delivery of cholesterol to the mitochondrial matrix where the first enzymatic reaction in the steroid hormone biosynthetic pathway occurs. A key response of adrenal cells to ACTH is activation of the cAMP-protein kinase A (PKA) signaling pathway. PKA activation results in an acute increase in expression and function of the Steroidogenic Acute Regulatory protein (StAR). StAR plays an essential role in steroidogenesis- it controls the hormone-dependent movement of cholesterol across the mitochondrial membranes. Currently StAR's mechanism of action remains a major unanswered question in the field. However, some insight may be gained from understanding the mechanism(s) controlling the PKA-dependent phosphorylation of StAR at S194/195 (mouse/human StAR), a modification that is required for function. This mini-review provides a background on StAR's biology with a focus on StAR phosphorylation. The model for StAR translation and phosphorylation at the outer mitochondrial membrane, the location for StAR function, is presented to highlight a unifying theme emerging from diverse studies. PMID:27999527

  2. HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

    PubMed Central

    Ramos-Montoya, Antonio; Lamb, Alastair D; Russell, Roslin; Carroll, Thomas; Jurmeister, Sarah; Galeano-Dalmau, Nuria; Massie, Charlie E; Boren, Joan; Bon, Helene; Theodorou, Vasiliki; Vias, Maria; Shaw, Greg L; Sharma, Naomi L; Ross-Adams, Helen; Scott, Helen E; Vowler, Sarah L; Howat, William J; Warren, Anne Y; Wooster, Richard F; Mills, Ian G; Neal, David E

    2014-01-01

    Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies. PMID:24737870

  3. Targeting RNA Splicing for Disease Therapy

    PubMed Central

    Havens, Mallory A.; Duelli, Dominik M.

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics. PMID:23512601

  4. Targeting RNA splicing for disease therapy.

    PubMed

    Havens, Mallory A; Duelli, Dominik M; Hastings, Michelle L

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics.

  5. Three-Dimensional Magnetic Restructuring in Two Homologous Solar Flares in the Seismically Active NOAA AR 11283

    NASA Technical Reports Server (NTRS)

    Liu, Chang; Deng, Na; Lee, Jeongwoo; Wiegelmann, Thomas; JIang, Chaowei; Dennis, Brian R.; Su, Yang; Donea, Alina; Wang, Haimin

    2014-01-01

    We carry out a comprehensive investigation comparing the three-dimensional magnetic field restructuring, flare energy release, and the helioseismic response of two homologous flares, the 2011 September 6 X2.1 (FL1) and September 7 X1.8 (FL2) flares in NOAA AR 11283. In our analysis, (1) a twisted flux rope (FR) collapses onto the surface at a speed of 1.5 km s(exp-1) after a partial eruption in FL1. The FR then gradually grows to reach a higher altitude and collapses again at 3 km s(exp-1) after a fuller eruption in FL2. Also, FL2 shows a larger decrease of the flux-weighted centroid separation of opposite magnetic polarities and a greater change of the horizontal field on the surface. These imply a more violent coronal implosion with corresponding more intense surface signatures in FL2. (2) The FR is inclined northward and together with the ambient fields, it undergoes a southward turning after both events. This agrees with the asymmetric decay of the penumbra observed in the peripheral regions. (3) The amounts of free magnetic energy and nonthermal electron energy released during FL1 are comparable to those of FL2 within the uncertainties of the measurements. (4) No sunquake was detected in FL1; in contrast, FL2 produced two seismic emission sources S1 and S2 both lying in the penumbral regions. Interestingly, S1 and S2 are connected by magnetic loops, and the stronger source S2 has a weaker vertical magnetic field. We discuss these results in relation to the implosion process in the low corona and the sunquake generation.

  6. Three-dimensional magnetic restructuring in two homologous solar flares in the seismically active NOAA AR 11283

    SciTech Connect

    Liu, Chang; Deng, Na; Lee, Jeongwoo; Wang, Haimin; Wiegelmann, Thomas; Jiang, Chaowei; Dennis, Brian R.; Su, Yang; Donea, Alina

    2014-11-10

    We carry out a comprehensive investigation comparing the three-dimensional magnetic field restructuring, flare energy release, and the helioseismic response of two homologous flares, the 2011 September 6 X2.1 (FL1) and September 7 X1.8 (FL2) flares in NOAA AR 11283. In our analysis, (1) a twisted flux rope (FR) collapses onto the surface at a speed of 1.5 km s{sup –1} after a partial eruption in FL1. The FR then gradually grows to reach a higher altitude and collapses again at 3 km s{sup –1} after a fuller eruption in FL2. Also, FL2 shows a larger decrease of the flux-weighted centroid separation of opposite magnetic polarities and a greater change of the horizontal field on the surface. These imply a more violent coronal implosion with corresponding more intense surface signatures in FL2. (2) The FR is inclined northward and together with the ambient fields, it undergoes a southward turning after both events. This agrees with the asymmetric decay of the penumbra observed in the peripheral regions. (3) The amounts of free magnetic energy and nonthermal electron energy released during FL1 are comparable to those of FL2 within the uncertainties of the measurements. (4) No sunquake was detected in FL1; in contrast, FL2 produced two seismic emission sources S1 and S2 both lying in the penumbral regions. Interestingly, S1 and S2 are connected by magnetic loops, and the stronger source S2 has a weaker vertical magnetic field. We discuss these results in relation to the implosion process in the low corona and the sunquake generation.

  7. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing.

  8. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  9. Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver*

    PubMed Central

    Walsh, Callee M.; Suchanek, Amanda L.; Cyphert, Travis J.; Kohan, Alison B.; Szeszel-Fedorowicz, Wioletta; Salati, Lisa M.

    2013-01-01

    Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing. PMID:23233666

  10. Serine arginine splicing factor 3 is involved in enhanced splicing of glucose-6-phosphate dehydrogenase RNA in response to nutrients and hormones in liver.

    PubMed

    Walsh, Callee M; Suchanek, Amanda L; Cyphert, Travis J; Kohan, Alison B; Szeszel-Fedorowicz, Wioletta; Salati, Lisa M

    2013-01-25

    Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.

  11. A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK).

    PubMed Central

    Yanagisawa, Ken; Tago, Kenji; Hayakawa, Morisada; Ohki, Motomichi; Iwahana, Hiroyuki; Tominaga, Shin-Ichi

    2003-01-01

    Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions. PMID:12418963

  12. Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors.

    PubMed Central

    Hanamura, A; Cáceres, J F; Mayeda, A; Franza, B R; Krainer, A R

    1998-01-01

    The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo. PMID:9630249

  13. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    SciTech Connect

    Lau, Aik Jiang; Chang, Thomas K.H.

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  14. A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

    PubMed Central

    Houman, Fariba; Rho, Seung Bae; Zhang, Jiansu; Shen, Xiaoyu; Wang, Chien-Chia; Schimmel, Paul; Martinis, Susan A.

    2000-01-01

    Mitochondrial leucyl-tRNA synthetase (LeuRS) in the yeast Saccharomyces cerevisiae provides two essential functions. In addition to aminoacylation, LeuRS functions in RNA splicing. The details of how it came to act in splicing are not known. Here we show that Mycobacterium tuberculosis and human mitochondrial LeuRSs can substitute in splicing for the S. cerevisiae mitochondrial LeuRS. Mutations of yeast mitochondrial LeuRS that had previously been shown to abolish splicing activity also eliminate splicing by the M. tuberculosis enzyme. These results suggest the role of LeuRS in splicing in yeast mitochondria results from features of the enzyme that are broadly conserved in evolution. These features are not likely to be designed for splicing per se, but instead have been adopted in yeast for that purpose. PMID:11087829

  15. Quantification of pre-mRNA escape rate and synergy in splicing

    PubMed Central

    Bonde, Marie Mi; Voegeli, Sylvia; Baudrimont, Antoine; Séraphin, Bertrand; Becskei, Attila

    2014-01-01

    Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks. PMID:25352554

  16. A high throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes

    PubMed Central

    Effenberger, Kerstin A.; Perriman, Rhonda J.; Bray, Walter M.; Lokey, R. Scott; Ares, Manuel; Jurica, Melissa S.

    2014-01-01

    The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing a myriad of subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high throughput assay for in vitro splicing using an RT-qPCR readout. In a pilot screen of 3,080 compounds we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly impact spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery. PMID:23771823

  17. The intronic splicing code: multiple factors involved in ATM pseudoexon definition.

    PubMed

    Dhir, Ashish; Buratti, Emanuele; van Santen, Maria A; Lührmann, Reinhard; Baralle, Francisco E

    2010-02-17

    Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5' splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.

  18. Identification and characterization of NF1 splicing mutations in Korean patients with neurofibromatosis type 1.

    PubMed

    Jang, Mi-Ae; Kim, Young-Eun; Kim, Sun Kyung; Lee, Myoung-Keun; Kim, Jong-Won; Ki, Chang-Seok

    2016-08-01

    Neurofibromatosis type I (NF1) is an autosomal dominant genetic disorder caused by NF1 mutations. Although mutations affecting mRNA splicing are the most common molecular defects in NF1, few studies have analyzed genomic DNA (gDNA)-mRNA correlations in Korean NF1 patients. In this study, we investigated 28 unrelated NF1 patients who showed splicing alterations in reverse transcription-PCR of NF1 mRNA and identified 24 different NF1 splicing mutations, 9 of which were novel. These mutations can be categorized into five groups: exon skipping resulting from mutations at authentic 5' and 3' splice sites (type I, 46%), cryptic exon inclusion caused by deep intronic mutations (type II, 8%), creation of new splice sites causing loss of exonic sequences (type III, 8%), activation of cryptic splice sites due to disruption of authentic splice sites (type IV, 25%) and exonic sequence alterations causing exon skipping (type V, 13%). In total, 42% of all splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites, making it difficult to identify the correct mutation sites at the gDNA level. These results add to the mutational spectrum of NF1 and further elucidate the gDNA-mRNA correlations of NF1 mutations.

  19. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

  20. The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

    PubMed

    Whisenant, Thomas C; Peralta, Eigen R; Aarreberg, Lauren D; Gao, Nina J; Head, Steven R; Ordoukhanian, Phillip; Williamson, Jamie R; Salomon, Daniel R

    2015-01-01

    Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA

  1. The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

    PubMed Central

    Aarreberg, Lauren D.; Gao, Nina J.; Head, Steven R.; Ordoukhanian, Phillip; Williamson, Jamie R.; Salomon, Daniel R.

    2015-01-01

    Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as “central” interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, “peripheral” interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly

  2. Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers. PMID:10022858

  3. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  4. Flight activity of USDA-ARS Russian honey bees (Hymenoptera: Apidae) during pollination of lowbush blueberries in Maine.

    PubMed

    Danka, Robert G; Beaman, Lorraine D

    2007-04-01

    Flight activity was compared in colonies of Russian honey bees, Apis mellifera L. (Hymenoptera: Apidae), and Italian bees during commercial pollination of lowbush blueberries (principally Vaccinium angustifolium Aiton) in Washington Co., ME, in late May and early June in 2003 and 2004. Colonies of the two stocks were managed equally in Louisiana during autumn through early spring preceding observations in late spring each year. Resulting average populations of adult bees and of brood were similar in colonies of the two bee stocks during pollination. Flight during pollination was monitored hourly on 6 d each year by counting bees exiting each colony per minute; counts were made manually with flight cones on 17 colonies per stock in 2003 and electronically with ApiSCAN-Plus counters on 20 colonies per stock in 2004. Analysis of variance showed that temperature, colony size (population of adult bees or brood), and the interaction of these effects were the strongest regulators of flight activity in both years. Russian and Italian bees had similar flight activity at any given colony size, temperature, or time of day. Flight increased linearly with rising temperatures and larger colony sizes. Larger colonies, however, were more responsive than smaller colonies across the range of temperatures measured. In 2003, flight responses to varying temperatures were less in the afternoon and evening (1500-1959 hours) than they were earlier in the day. Russian colonies had flight activity that was suitable for late spring pollination of lowbush blueberries.

  5. Oncogenes and RNA splicing of human tumor viruses.

    PubMed

    Ajiro, Masahiko; Zheng, Zhi-Ming

    2014-09-01

    Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis.

  6. Regulation of AR Degradation and Function by Ubiquitylation

    DTIC Science & Technology

    2015-10-01

    assays to examine effects of site directed mutants on AR transcriptional activity. Lentiviruses encoding AR wild-type or mutants were used to establish...regulated Luciferase reporter gene then showed that only the K911A mutant AR had increased activity (~2-fold) (Fig. 1A). Table 1. AR...the stably expressed AR WT versus K911R mutant . As shown in figure 1B, the K911R mutant had increased activity in response to 1 nM DHT. Finally

  7. A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence.

    PubMed

    Hovhannisyan, Ruben H; Carstens, Russ P

    2005-01-01

    Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb. This element promoted inclusion of exon IIIb while at the same time silencing exon IIIc inclusion in cells expressing FGFR2-IIIb; hence, we have termed this element ISE/ISS-3 (for "intronic splicing enhancer-intronic splicing silencer 3"). Silencing of exon IIIc splicing by ISE/ISS-3 was shown to require a branch point sequence (BPS) using G as the primary branch nucleotide. Replacing a consensus BPS with A as the primary branch nucleotide resulted in constitutive splicing of exon IIIc. Our results suggest that the branch point sequence constitutes an important component that can contribute to the efficiency of exon definition of alternatively spliced cassette exons. Noncanonical branch points may thus facilitate cell-type-specific silencing of regulated exons by flanking cis elements.

  8. 40Ar/39Ar ages of the AD 79 eruption of Vesuvius, Italy

    USGS Publications Warehouse

    Lanphere, Marvin A.; Champion, Duane E.; Melluso, Leone; Morra, Vincenzo; Perrotta, Annamaria; Scarpati, Claudio; Tedesco, Dario; Calvert, Andrew T.

    2007-01-01

    The Italian volcano, Vesuvius, erupted explosively in AD 79. Sanidine from pumice collected at Casti Amanti in Pompeii and Villa Poppea in Oplontis yielded a weighted-mean 40Ar/39Ar age of 1925±66 years in 2004 (1σ uncertainty) from incremental-heating experiments of eight aliquants of sanidine. This is the calendar age of the eruption. Our results together with the work of Renne et al. (1997) and Renne and Min (1998) demonstrate the validity of the 40Ar/39Ar method to reconstruct the recent eruptive history of young, active volcanoes.

  9. 40Ar/39Ar ages of the AD 79 eruption of Vesuvius, Italy

    NASA Astrophysics Data System (ADS)

    Lanphere, Marvin; Champion, Duane; Melluso, Leone; Morra, Vincenzo; Perrotta, Annamaria; Scarpati, Claudio; Tedesco, Dario; Calvert, Andrew

    2007-01-01

    The Italian volcano, Vesuvius, erupted explosively in AD 79. Sanidine from pumice collected at Casti Amanti in Pompeii and Villa Poppea in Oplontis yielded a weighted-mean 40Ar/39Ar age of 1925±66 years in 2004 (1σ uncertainty) from incremental-heating experiments of eight aliquants of sanidine. This is the calendar age of the eruption. Our results together with the work of Renne et al. (1997) and Renne and Min (1998) demonstrate the validity of the 40Ar/39Ar method to reconstruct the recent eruptive history of young, active volcanoes.

  10. Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences.

    PubMed

    Lipscombe, Diane; Andrade, Arturo; Allen, Summer E

    2013-07-01

    Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal Ca(V) channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinson's disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of Ca(V) channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of Ca(V) channel structures and functions. The precise composition of Ca(V) channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of Ca(V) splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of Ca(V) pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels.

  11. Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues.

    PubMed

    Amitai, Gil; Dassa, Bareket; Pietrokovski, Shmuel

    2004-01-30

    Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.

  12. Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

    PubMed Central

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-01-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  13. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  14. Evolutionary conservation of alternative splicing in chicken

    PubMed Central

    Katyal, S.; Gao, Z.; Liu, R.-Z.; Godbout, R.

    2013-01-01

    Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

  15. Alcoholism and alternative splicing of candidate genes.

    PubMed

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  16. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

  17. Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

    PubMed

    Venables, Julian P; Vignal, Emmanuel; Baghdiguian, Stephen; Fort, Philippe; Tazi, Jamal

    2012-01-01

    Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue

  18. Conserved RNA secondary structures promote alternative splicing.

    PubMed

    Shepard, Peter J; Hertel, Klemens J

    2008-08-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site strength, splicing regulators, the exon/intron architecture, and the process of pre-mRNA synthesis itself. RNA secondary structures have also been proposed to influence alternative splicing as stable RNA secondary structures that mask splice sites are expected to interfere with splice-site recognition. Using structural and functional conservation, we identified RNA structure elements within the human genome that associate with alternative splice-site selection. Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease.

  19. Development of AR-V7 as a putative treatment selection marker for metastatic castration-resistant prostate cancer.

    PubMed

    Luo, Jun

    2016-01-01

    Prostate cancer cells demonstrate a remarkable "addiction" to androgen receptor (AR) signaling in all stages of disease progression. As such, suppression of AR signaling remains the therapeutic goal in systemic treatment of prostate cancer. A number of molecular alterations arise in patients treated with AR-directed therapies. These molecular alterations may indicate the emergence of treatment resistance and may be targeted for the development of novel agents for prostate cancer. The presence of functional androgen receptor splice variants may represent a potential explanation for resistance to abiraterone and enzalutamide, newer AR-directed agents developed to treat metastatic castration-resistant prostate cancer (mCRPC). In the last 8 years, many androgen receptor splice variants have been identified and characterized. Among these, androgen receptor splice variant-7 (AR-V7) has been investigated extensively. In AR-V7, the entire COOH-terminal ligand-binding domain of the canonical AR is truncated and replaced with a variant-specific peptide of 16 amino acids. Functionally, AR-V7 is capable of mediating constitutive nuclear localization and androgen receptor signaling in the absence of androgens, or in the presence of enzalutamide. In this review, we will focus on clinical translational studies involving detection/measurement of AR-V7. Methods have been developed to detect AR-V7 in clinical mCRPC specimens. AR-V7 can be reliably measured in both tissue and circulating tumor cells derived from mCRPC patients, making it possible to conduct both cross-sectional and longitudinal clinical correlative studies. Current evidence derived from studies focusing on detection of AR-V7 in mCRPC support its potential clinical utility as a treatment selection marker.

  20. In Vivo Analysis of Alternative Pre-mRNA Splicing.

    DTIC Science & Technology

    1996-10-01

    SF2 influences 5’ splice site selection by activating proximal sites. Cell 62: 35-42 Koehorst SG, Jacobs HM, Thijssen JH, Blankenstein MA (1993) Wild...S, Frati L and Gulino A (1995) Estrogen receptors: new perspectives in breast cancer management. J. Ster. Bioch . Mol. Biol. 49:327-331. Pfeffer U

  1. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    PubMed Central

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks. PMID:24101931

  2. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  3. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  4. DBASS3 and DBASS5: databases of aberrant 3'- and 5'-splice sites.

    PubMed

    Buratti, Emanuele; Chivers, Martin; Hwang, Gyulin; Vorechovsky, Igor

    2011-01-01

    DBASS3 and DBASS5 provide comprehensive repositories of new exon boundaries that were induced by pathogenic mutations in human disease genes. Aberrant 5'- and 3'-splice sites were activated either by mutations in the consensus sequences of natural exon-intron junctions (cryptic sites) or elsewhere ('de novo' sites). DBASS3 and DBASS5 currently contain approximately 900 records of cryptic and de novo 3'- and 5'-splice sites that were produced by over a thousand different mutations in approximately 360 genes. DBASS3 and DBASS5 data can be searched by disease phenotype, gene, mutation, location of aberrant splice sites in introns and exons and their distance from authentic counterparts, by bibliographic references and by the splice-site strength estimated with several prediction algorithms. The user can also retrieve reference sequences of both aberrant and authentic splice sites with the underlying mutation. These data will facilitate identification of introns or exons frequently involved in aberrant splicing, mutation analysis of human disease genes and study of germline or somatic mutations that impair RNA processing. Finally, this resource will be useful for fine-tuning splice-site prediction algorithms, better definition of auxiliary splicing signals and design of new reporter assays. DBASS3 and DBASS5 are freely available at http://www.dbass.org.uk/.

  5. Mammalian mRNA Splice-Isoform Selection Is Tightly Controlled

    PubMed Central

    Chisa, Jennifer L.; Burke, David T.

    2007-01-01

    Post-transcriptional RNA processing is an important regulatory control mechanism for determining the phenotype of eukaryotic cells. The processing of a transcribed RNA species into alternative splice isoforms yields products that can perform different functions. Each type of cell in a multi-cellular organism is presumed to actively control the relative quantities of alternative splice isoforms. In this study, the alternatively spliced isoforms of five mRNA transcription units were examined by quantitative reverse transcription–PCR amplification. We show that interindividual variation in splice-isoform selection is very highly constrained when measured in a large population of genetically diverse mice (i.e., full siblings; N = 150). Remarkably, splice-isoform ratios are among the most invariant phenotypes measured in this population and are confirmed in a second, genetically distinct population. In addition, the patterns of splice-isoform selection show tissue-specific and age-related changes. We propose that splice-isoform selection is exceptionally robust to genetic and environmental variability and may provide a control point for cellular homeostasis. As a consequence, splice-isoform ratios may be useful as a practical quantitative measure of the physiological status of cells and tissues. PMID:17179090

  6. Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy

    PubMed Central

    Chen, Jieliang; Wu, Min; Wang, Fan; Zhang, Wen; Wang, Wei; Zhang, Xiaonan; Zhang, Jiming; Liu, Yinghui; Liu, Yi; Feng, Yanling; Zheng, Ye; Hu, Yunwen; Yuan, Zhenghong

    2015-01-01

    During hepatitis B virus (HBV) replication, spliced HBV genomes and splice-generated proteins have been widely described, however, their biological and clinical significance remains to be defined. Here, an elevation of the proportion of HBV spliced variants in the sera of patients with chronic hepatitis B (CHB) is shown to correlate with an impaired respond to interferon-α (IFN-α) therapy. Transfection of the constructs encoding the three most dominant species of spliced variants into cells or ectopic expression of the two major spliced protein including HBSP and N-terminal-truncated viral polymerase protein result in strong suppression of IFN-α signaling transduction, while mutation of the major splicing-related sites of HBV attenuates the viral anti-IFN activities in both cell and mouse models. These results have associated the productions of HBV spliced variants with the failure response to IFN therapy and illuminate a novel mechanism where spliced viral products are employed to resist IFN-mediated host defense. PMID:26585041

  7. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  8. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  9. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  10. Plasma Motions and Turbulent Magnetic Diffusivity of Active Region AR 12158 Using a Minimum Energy Functional and Non-Force-Free Reconstructions of Vector Magnetograms

    NASA Astrophysics Data System (ADS)

    Tremblay, Benoit; Vincent, Alain

    2017-01-01

    We present a generalization of the resistive minimum-energy fit (MEF-R: Tremblay and Vincent, Solar Phys. 290, 437, 2015) for non-force-free (NFF) magnetic fields. In MEF-R, an extremum principle is used to infer two-dimensional maps of plasma motions [boldsymbol{v}(x,y)] and magnetic eddy diffusivity [η _{eddy}(x,y)] at the photosphere. These reconstructions could be used as boundary conditions in data-driven simulations or in data assimilation. The algorithm is validated using the analytical model of a resistive expanding spheromak by Rakowski, Laming, and Lyutikov ( Astrophys. J. 730, 30, 2011). We study the flaring Active Region AR 12158 using a series of magnetograms and Dopplergrams provided by the Helioseismic and Magnetic Imager (HMI) onboard the Solar Dynamics Observatory (SDO). The results are discussed for a non-force-free magnetic-field reconstruction [boldsymbol{B}_{NFF}] (Hu and Dasgupta in Solar Phys. 247, 87, 2008). We found that the vertical plasma velocities [vz(x,y)] inferred using MEF-R are very similar to the observed Doppler velocities [vr(x,y)]. Finally, we study the potential spatial correlation between microturbulent velocities and significant values of η_{eddy}(x,y).

  11. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  12. Trans-splicing with the group I intron ribozyme from Azoarcus

    PubMed Central

    Dolan, Gregory F.; Müller, Ulrich F.

    2014-01-01

    Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNAIle group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5′ terminus. In contrast to the previously optimized 5′-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5′ portion and the ribozyme 3′ exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme. PMID:24344321

  13. A novel variant of the putative demethylase gene, s-JMJD1C, is a coactivator of the AR.

    PubMed

    Wolf, Siegmund S; Patchev, Vladimir K; Obendorf, Maik

    2007-04-01

    Evidence is accumulating in support of the view that tissue-specific effects of steroid hormones depend on the recruitment of nuclear receptor comodulator proteins. The latter interact directly with the hormone receptors and modify their transcriptional effects on specific target genes. The mechanisms of comodulator influence on nuclear receptor-controlled gene transcription is only partially understood. Here, we describe the discovery of a new AR coactivator which belongs to the JmjC containing enzyme family as a novel variant of JMJD1C (jumonji domain-containing 1C). By using a fragment of the human AR (aa 325-919) as bait in a yeast two-hybrid screen, a region of the human JMJD1C gene was identified as interacting with AR. A novel splice variant s-JMJD1C was amplified by RACE, and the binding to AR was analysed by GST-pull-down and mammalian one-hybrid experiments. As a nuclear-localized protein, the s-JMJD1C gene is expressed in a variety of human tissues. In the brain, this protein is present in several, but not confined to, AR-expressing neuronal populations and its abundance varies with the hormonal status in a region-specific fashion. Interestingly, the expression of s-JMJD1C is reduced in breast cancer tumors and significantly higher in normal breast tissues indicating a putative role in tumor suppression. As s-JMJD1C has putative demethylase activity, removal of methylation seems to be important for nuclear receptor-based gene regulation.

  14. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.

  15. 40Ar/39Ar geochronological constraints on the formation of the Dayingezhuang gold deposit: New implications for timing and duration of hydrothermal activity in the Jiaodong gold province, China

    USGS Publications Warehouse

    Yang, Li-Qiang; Deng, J.; Goldfarb, Richard J.; Zhang, Jiahua; Gao, Bang-Fei; Wang, Zhong-Liang

    2014-01-01

    China's largest gold resource is located in the highly endowed northwestern part of the Jiaodong gold province. Most gold deposits in this area are associated with the NE- to NNE-trending shear zones on the margins of the 130–126 Ma Guojialing granite. These deposits collectively formed at ca. 120 ± 5 Ma during rapid uplift of the granite. The Dayingezhuang deposit is a large (> 120 t Au) orogenic gold deposit in the same area, but located along the eastern margin of the Late Jurassic Linglong Metamorphic Core Complex. New 40Ar/39Ar geochronology on hydrothermal sericite and muscovite from the Dayingezhuang deposit indicate the gold event is related to evolution of the core complex at 130 ± 4 Ma and is the earliest important gold event that is well-documented in the province. The Dayingezhuang deposit occurs along the Linglong detachment fault, which defines the eastern edge of the ca. 160–150 Ma Linglong granite–granodiorite massif. The anatectic rocks of the massif were rapidly uplifted, at rates of at least 1 km/m.y. from depths of 25–30 km, to form the metamorphic core complex. The detachment fault, with Precambrian metamorphic basement rocks in the hangingwall and the Linglong granitoids and migmatites in the footwall, is characterized by early mylonitization and a local brittle overprinting in the footwall. Gold is associated with quartz–sericite–pyrite–K-feldspar altered footwall cataclasites at the southernmost area of the brittle deformation along the detachment fault. Our results indicate that there were two successive, yet distinct gold-forming tectonic episodes in northwestern Jiaodong. One event first reactivated the detachment fault along the edge of the Linglong massif between 134 and 126 Ma, and then a second reactivated the shears along the margins of the Guojialing granite. Both events may relate to a component of northwest compression after a middle Early Cretaceous shift from regional NW–SE extension to a NE

  16. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11.

    PubMed

    Heintz, Caroline; Dobrowolski, Steven F; Andersen, Henriette Skovgaard; Demirkol, Mübeccel; Blau, Nenad; Andresen, Brage Storstein

    2012-08-01

    In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.

  17. The cistrome and gene signature of androgen receptor splice variants in castration-resistant prostate cancer cells

    PubMed Central

    Lu, Ji; Lonergan, Peter E.; Nacusi, Lucas P.; Wang, Liguo; Schmidt, Lucy J.; Sun, Zhifu; Van der Steen, Travis; Boorjian, Stephen A.; Kosari, Farhad; Vasmatzis, George; Klee, George G.; Balk, Steven P.; Huang, Haojie; Wang, Chunxi; Tindall, Donald J.

    2015-01-01

    Purpose Spliced variant forms of androgen receptor (AR-Vs) have been identified recently in castration-resistant prostate cancer (CRPC) cell lines and clinical samples. We identified the cistrome and gene signature of AR-Vs in CRPC cell lines and determined the clinical significance of AR-Vs regulated genes. Materials and Methods The CRPC cell line 22Rv1, which expresses both full length androgen receptor (AR-FL) and AR-Vs endogenously, was used as the research model. We established 22Rv1-ARFL−/ARVs+ and 22Rv1- ARFL−/ARVs− through RNA interference. ChIP-seq and microarray techniques were used to identify cistrome and gene expression profiles of AR-Vs in the absence of androgen. Results AR-Vs binding sites were identified in 22Rv1-ARFL−/ARVs+. A set of genes was found to be regulated uniquely by AR-Vs, but not by full-length AR in the absence of androgen. Integrated analysis revealed that some genes are modulated by AR-Vs directly. Unsupervised clustering analysis showed that the AR-Vs gene signature can differentiate not only benign from malignant prostate tissue, but also localized prostate cancer from metastatic CRPC specimens. Some genes that were modulated uniquely by AR-Vs also correlated with histological grade and biochemical failure. Conclusions We conclude that AR-Vs can bind to DNA independently of full-length AR in the absence of androgen and modulate a unique set of genes that is not regulated by AR-FL. The AR-Vs gene signature correlates with disease progression, and distinguishes between primary cancer and CRPC specimens, as well as benign and malignant prostate specimens. PMID:25132238

  18. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

    PubMed Central

    Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

  19. 39Ar- 40Ar ages of martian nakhlites

    NASA Astrophysics Data System (ADS)

    Park, Jisun; Garrison, Daniel H.; Bogard, Donald D.

    2009-04-01

    We report 39Ar- 40Ar ages of whole rock (WR) and plagioclase and pyroxene mineral separates of nakhlites MIL 03346 and Y-000593, and of WR samples of nakhlites NWA 998 and Nakhla. All age spectra are complex and indicate variable degrees of 39Ar recoil and variable amounts of trapped 40Ar in the samples. Thus, we examine possible Ar-Ar ages in several ways. From consideration of both limited plateau ages and isochron ages, we prefer Ar-Ar ages of NWA 998 = 1334 ± 11 Ma, MIL 03346 = 1368 ± 83 Ma (mesostasis) and 1334 ± 54 Ma (pyroxene), Y-000593 = 1367 ± 7 Ma, and Nakhla = 1357 ± 11 Ma, (2 σ errors). For NWA 998 and MIL 03346 the Ar-Ar ages are within uncertainties of preliminary Rb-Sr isochron ages reported in the literature. These Ar-Ar ages for Y-000593 and Nakhla are several Ma older than Sm-Nd ages reported in the literature. We conclude that the major factor in producing Ar-Ar ages slightly too old is the presence of small amounts of trapped martian or terrestrial 40Ar on weathered grain surfaces that was degassed along with the first several percent of 39Ar. A total K- 40Ar isochron for WR and mineral data from five nakhlites analyzed by us, plus Lafayette data in the literature, gives an isochron age of 1325 ± 18 Ma (2 σ). We emphasize the precision of this isochron over the value of the isochron age. Our Ar-Ar data are consistent with a common formation age for nakhlites. The cosmic-ray exposure (CRE) age for NWA 998 of ˜12 Ma is also similar to CRE ages for other nakhlites.

  20. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  1. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  2. Homologous SV40 RNA trans-splicing: a new mechanism for diversification of viral sequences and phenotypes.

    PubMed

    Eul, Joachim; Patzel, Volker

    2013-11-01

    Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5' donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5'ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5'ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes.

  3. Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

    PubMed

    Sewer, Marion B; Nguyen, Viet Q; Huang, Ching-Jung; Tucker, Philip W; Kagawa, Norio; Waterman, Michael R

    2002-04-01

    The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

  4. Registered report: androgen receptor splice variants determine taxane sensitivity in prostate cancer.

    PubMed

    Shan, Xiaochuan; Danet-Desnoyers, Gwenn; Fung, Juan José; Kosaka, Alan H; Tan, Fraser; Perfito, Nicole; Lomax, Joelle; Iorns, Elizabeth

    2015-01-01

    The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

  5. Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

    PubMed

    Zhang, Qiao; Kota, Krishna P; Alam, Samer G; Nickerson, Jeffrey A; Dickinson, Richard B; Lele, Tanmay P

    2016-06-01

    Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

  6. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

    PubMed Central

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-01-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations. PMID:21407258

  7. Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer

    PubMed Central

    Jones, Dominic; Noble, Martin; Wedge, Steve R.; Robson, Craig N.; Gaughan, Luke

    2017-01-01

    Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC. PMID:28205582

  8. Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer.

    PubMed

    Jones, Dominic; Noble, Martin; Wedge, Steve R; Robson, Craig N; Gaughan, Luke

    2017-02-16

    Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC.

  9. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  10. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

  11. Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

    PubMed Central

    Carpenter, Beth M.; West, Abby L.; Gancz, Hanan; Servetas, Stephanie L.; Pich, Oscar Q.; Gilbreath, Jeremy J.; Hallinger, Daniel R.; Forsyth, Mark H.; Merrell, D. Scott; Michel, Sarah L. J.

    2015-01-01

    Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct “cross-talk” between HpArsRS and HpNikR at

  12. Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription.

    PubMed

    Carpenter, Beth M; West, Abby L; Gancz, Hanan; Servetas, Stephanie L; Pich, Oscar Q; Gilbreath, Jeremy J; Hallinger, Daniel R; Forsyth, Mark H; Merrell, D Scott; Michel, Sarah L J

    2015-01-01

    Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at

  13. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.

  14. Splicing of intron 3 of human BACE requires the flanking introns 2 and 4.

    PubMed

    Annies, Maik; Stefani, Muriel; Hueber, Andreas; Fischer, Frauke; Paganetti, Paolo

    2009-10-16

    Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer's disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.

  15. The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

    PubMed

    Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

    2015-05-01

    When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

  16. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  17. MEIS1 functions as a potential AR negative regulator

    SciTech Connect

    Cui, Liang; Yang, Yutao; Hang, Xingyi; Cui, Jiajun; Gao, Jiangping

    2014-10-15

    The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein–protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostate specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor. - Highlights: • A potential interaction was identified between MEIS1 and AR signaling. • Overexpression of MEIS1 reduced the expression of AR target gene. • MEIS1 modulated AR cytoplasm/nucleus translocation. • MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells.

  18. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

  19. Modulation of Stat3 Alternative Splicing in Breast Cancer

    DTIC Science & Technology

    2010-09-01

    The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an...Stat3 Alternative Splicing in Breast Cancer Dr. Luca Cartegni Sloan-Kettering Institute New York, NY 10021 Stat3 is a transcription factor...constitutively active in a large number of breast cancers and other tumors, where it works as a central player in the activation of multiple oncogenic pathways

  20. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html.

  1. Ar/Ar Dating Independent of Monitor Standard Ages

    NASA Astrophysics Data System (ADS)

    Boswell, S.; Hemming, S. R.

    2015-12-01

    Because the reported age of an analyzed sample is dependent on the age of the co-irradiated monitor standard(s), Ar/Ar dating is a relative dating technique. There is disagreement at the 1% scale in the age of commonly used monitor standards, and there is a great need to improve the inter-laboratory calibrations. Additionally, new approaches and insights are needed to meet the challenge of bringing the Ar/Ar chronometer to the highest possible precision and accuracy. In this spirit, we present a conceptual framework for Ar/Ar dating that does not depend on the age of monitor standards, but only on the K content of a solid standard. The concept is demonstrated by introducing a re-expressed irradiation parameter (JK) that depends on the ratio of 39ArK to 40Ar* rather than the 40Ar*/39ArK ratio. JK is equivalent to the traditional irradiation parameter J and is defined as JK = (39Ar/40K) • (λ/λe). The ultimate precision and accuracy of the method will depend on how precisely and accurately the 39Ar and 40K can be estimated, and will require isotope dilution measurements of both from the same aliquot. We are testing the workability of our technique at the 1% level by measuring weighed and irradiated hornblende and biotite monitor standards using GLO-1 glauconite to define a calibration curve for argon signals versus abundance.

  2. RBFOX1 Cooperates with MBNL1 to Control Splicing in Muscle, Including Events Altered in Myotonic Dystrophy Type 1

    PubMed Central

    Klinck, Roscoe; Fourrier, Angélique; Thibault, Philippe; Toutant, Johanne; Durand, Mathieu; Lapointe, Elvy; Caillet-Boudin, Marie-Laure; Sergeant, Nicolas; Gourdon, Geneviève; Meola, Giovanni; Furling, Denis; Puymirat, Jack; Chabot, Benoit

    2014-01-01

    With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1. PMID:25211016

  3. Arsenic resistance strategy in Pantoea sp. IMH: Organization, function and evolution of ars genes

    PubMed Central

    Wang, Liying; Zhuang, Xuliang; Zhuang, Guoqiang; Jing, Chuanyong

    2016-01-01

    Pantoea sp. IMH is the only bacterium found in genus Pantoea with a high As resistance capacity, but its molecular mechanism is unknown. Herein, the organization, function, and evolution of ars genes in IMH are studied starting with analysis of the whole genome. Two ars systems - ars1 (arsR1B1C1H1) and ars2 (arsR2B2C2H2) - with low sequence homology and two arsC-like genes, were found in the IMH genome. Both ars1 and ars2 are involved in the As resistance, where ars1 is the major contributor at 15 °C and ars2 at 30 °C. The difference in the behavior of these two ars systems is attributed to the disparate activities of their arsR promoters at different temperatures. Sequence analysis based on concatenated ArsRBC indicates that ars1 and ars2 clusters may be acquired from Franconibacter helveticus LMG23732 and Serratia marcescens (plasmid R478), respectively, by horizontal gene transfer (HGT). Nevertheless, two arsC-like genes, probably arising from the duplication of arsC, do not contribute to the As resistance. Our results indicate that Pantoea sp. IMH acquired two different As resistance genetic systems by HGT, allowing the colonization of changing ecosystems, and highlighting the flexible adaptation of microorganisms to resist As. PMID:27966630

  4. Sugar beet activities of the USDA-ARS East Lansing conducted in cooperation with Saginaw Valley Bean and Beet Farm during 2011 (including Project 905)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation and rating plots were planted at the Saginaw Valley Research & Extension Center in Frankenmuth, MI in 2011 that focused on Cercospora leaf spot performance, conducted in conjunction with Beet Sugar Development Foundation and including USDA-ARS cooperators. 263 breeding lines were tested i...

  5. Sugar Beet Activities of the USDA-ARS East Lansing Conducted in Cooperation with Saginaw Valley Bean and Beet Farm During 2009

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two evaluation plots were planted at the Saginaw Valley Research & Extension Center in Frankenmuth, MI in 2009; one agronomic trial and one combined Cercospora evaluation trial. All trials were planted, following normal fall and spring tillage operations, with a USDA-ARS modified John Deere/Almaco ...

  6. Hydrogen peroxide triggers a novel alternative splicing of arsenic (+3 oxidation state) methyltransferase gene.

    PubMed

    Sumi, Daigo; Takeda, Chieri; Yasuoka, Daiki; Himeno, Seiichiro

    2016-11-04

    We previously reported that two splicing variants of human AS3MT mRNA, exon-3 skipping form (Δ3) and exons-4 and -5 skipping form (Δ4,5), were detected in HepG2 cells and that both variants lacked arsenic methylation activity (Sumi et al., 2011). Here we studied whether hydrogen peroxide (H2O2) triggers alternative splicing of AS3MT mRNA. The results showed that exposure of HepG2 cells to H2O2 resulted in increased levels of a novel spliced form skipping exon-3 to exon-10 (Δ3-10) in an H2O2-concentration-dependent manner, although no change was detected in the mRNA levels of Δ3 AS3MT. We found decreased protein levels of serine/arginine-rich 40 (SRp40), which we determined to be a candidate splice factor for controlling the splicing of AS3MT mRNA. We next compared the amounts of methylated arsenic metabolites between control and H2O2-exposed HepG2 cells after the addition of arsenite as a substance. The results showed lower levels of methylated arsenic metabolites in HepG2 cells exposed to H2O2. These data suggest that the splicing of AS3MT pre-mRNA was disconcerted by oxidative stress and that abnormal alternative splicing of AS3MT mRNA may affect arsenic methylation ability.

  7. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    PubMed

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

  8. An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons in the mouse hippocampus

    PubMed Central

    Nguyen, Thi-Minh; Schreiner, Dietmar; Xiao, Le; Traunmüller, Lisa; Bornmann, Caroline; Scheiffele, Peter

    2016-01-01

    The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit function DOI: http://dx.doi.org/10.7554/eLife.22757.001 PMID:27960072

  9. Computational Methods and Correlation of Exon-skipping Events with Splicing, Transcription, and Epigenetic Factors

    PubMed Central

    Wang, Jianbo; Ye, Zhenqing; Huang, Tim H.; Shi, Huidong; Jin, Victor X.

    2016-01-01

    Alternative splicing is widely recognized for playing roles in regulating genes and creating gene diversity. Consequently the identification and quantification of differentially spliced transcripts are pivotal for transcriptome analysis. However, how these diversified isoforms are spliced during genomic transcription and protein expression and what biological factors might influence the regulation of this are still required for further exploration. The advances in next-generation sequencing of messenger RNA (RNA-seq) have enabled us to survey gene expression and splicing more accurately. We have introduced a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium, and integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. In a separate study, we also apply this algorithm in prostate cancer in The Cancer Genomics Atlas (TCGA) for de novo skipping event discovery to the understanding of abnormal splicing in each patient and to identify potential markers for prediction and progression of diseases. PMID:27807836

  10. A feedback loop regulates splicing of the spinal muscular atrophy-modifying gene, SMN2.

    PubMed

    Jodelka, Francine M; Ebert, Allison D; Duelli, Dominik M; Hastings, Michelle L

    2010-12-15

    Spinal muscular atrophy (SMA) is a neurological disorder characterized by motor neuron degeneration and progressive muscle paralysis. The disease is caused by a reduction in survival of motor neuron (SMN) protein resulting from homozygous deletion of the SMN1 gene. SMN protein is also encoded by SMN2. However, splicing of SMN2 exon 7 is defective, and consequently, the majority of the transcripts produce a truncated, unstable protein. SMN protein itself has a role in splicing. The protein is required for the biogenesis of spliceosomal snRNPs, which are essential components of the splicing reaction. We now show that SMN protein abundance affects the splicing of SMN2 exon 7, revealing a feedback loop inSMN expression. The reduced SMN protein concentration observed in SMA samples and in cells depleted of SMN correlates with a decrease in cellular snRNA levels and a decrease in SMN2 exon 7 splicing. Furthermore, altering the relative abundance or activity of individual snRNPs has distinct effects on exon 7 splicing, demonstrating that core spliceosomal snRNPs influence SMN2 alternative splicing. Our results identify a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein. These results imply that a modest increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for assessing the therapeutic potential of SMA treatments and understanding disease pathogenesis.

  11. Unfolding the mystery of alternative splicing through a unique method of in vivo selection.

    PubMed

    Singh, Ravindra N

    2007-05-01

    Alternative splicing of pre-messenger RNA (pre-mRNA) is a fundamental mechanism of gene regulation in higher eukaryotes. In addition to creating protein diversity, alternative splicing provides the safest mode of gene evolution. Of late, more and more forms of alternatively spliced transcripts (mRNAs) are being discovered for key genes. Some of the alternatively spliced transcripts are also associated with major human diseases. This has created a sense of urgency to find the methods by which regulation of alternative splicing of specific exons could be best understood. Here I review a powerful in vivo selection method that uses a combinatorial library of partially random sequences. Several advantages of this method include in vivo analysis of large sequences, identification of unique sequence motifs, determination of relative strength of splice sites and identification of long-distance interactions including role of RNA structures. This unique method could be applied to identify tissue-specific cis-elements. Similarly, the method is suitable to find cis-elements that become active in response to specific treatments of cells. Considering this unbiased method uses in vivo conditions, it has potential to identify critical regulatory elements as therapeutic targets for a growing number of splicing-associated diseases.

  12. Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

    PubMed

    Iijima, Takatoshi; Hidaka, Chiharu; Iijima, Yoko

    2016-08-01

    Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases.

  13. The RNA-binding protein QKI suppresses cancer-associated aberrant splicing.

    PubMed

    Zong, Feng-Yang; Fu, Xing; Wei, Wen-Juan; Luo, Ya-Ge; Heiner, Monika; Cao, Li-Juan; Fang, Zhaoyuan; Fang, Rong; Lu, Daru; Ji, Hongbin; Hui, Jingyi

    2014-04-01

    Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.

  14. 40Ar/39Ar Dating of Volcanic Glass

    NASA Astrophysics Data System (ADS)

    Morgan, L. E.; Renne, P. R.; Watkins, J. M.

    2007-12-01

    Application of the 40Ar/39Ar method to volcanic glasses has been somewhat stigmatized following several studies demonstrating secondary mobility of K and Ar. Much of the stigma is unwarranted, however, since most studies only impugned the reliability of the K-Ar and 40Ar/39Ar techniques when applied to glass shards rather than obsidian clasts with low surface area to volume ratios. We provide further evidence for problematic K loss and/or 39Ar recoil ejection from glass shards in 40Ar/39Ar step heating results for comagmatic feldspars and shards. In an extreme case, the plateau age of the feldspars (0.17 ± 0.03 Ma at 2σ) is significantly younger than the plateau age of the glass (0.85 ± 0.05 Ma at 2σ). If the feldspar age is reasonably interpreted as the eruption age of the ash, it is likely that the glass shards experienced K and/or 39Ar loss. Electron microprobe analyses of the glass shards have low totals (~93%) and no systematic lateral variability (i.e., diffusion gradients) in K, suggesting that the lengthscale of the glass shards is smaller than the lengthscale of K diffusion. Obsidian clasts should not be as susceptible to K loss since any hydrated (K-depleted) volume represents a small fraction of the total material and can often be physically removed prior to analysis. Samples described here are detrital obsidian clasts from the Afar region of Ethiopia. Evidence from Fourier Transform Infrared Spectroscopy (FTIR), and previous work by Anovitz (1999), confirm that the scale of water and potassium mobility are often small in comparison to the size of obsidian clasts but large enough to effect the bulk composition of glass shards. This expectation is confirmed in another tuff wherein comagmatic obsidian clasts and sanidine phenocrysts yield indistinguishable 40Ar/39Ar ages of 4.4 Ma High abundances of non-radiogenic 40Ar, and kinetic fractionation of Ar isotopes during quenching and/or laboratory degassing resulting in incomplete equilibration between

  15. Combined K-Ar and 40Ar/39Ar dating of the upper Jaramillo reversal

    NASA Astrophysics Data System (ADS)

    Guillou, Herve; Carracedo, Juan Carlos; Kissel, Catherine; Laj, Carlo; Nomade, Sebastien; Perez Torrado, Francisco Jose; Rodriguez Gonzalez, Alejandro; Wandres, Camille

    2013-04-01

    The Jaramillo subchron was first evidenced in 1966 (Doell and Dalrymple) through the Rhyolotic domes of the Valles Caldera, New Mexico (USA). 40Ar/39Ar studies achieved by Spell et McDougall (1992), Spell et Harrison (1993), Izett and Obradovich (1994) and Singer et al. (1994) defined the base of this subchron at 1053±6 ka, and the ceiling at 986±5 ka. Channell et al. (2009) delimited the age of the Jaramillo subchron by astronomic calibration (base 1071 ka, top 990 ka). To provide additional absolute ages on this geomagnetic period, which is critical to improve our knowledge of the earth magnetic field behaviour, we have carried out a study combining paleomagnetism and isotopic dating of a lava sequence from Tenerife island. This sequence of basaltic lava flows is some 500 m thick. The first 400 m present, based on field magnetometer measurements, normal polarity lavas, with dykes of normal and reverse polarity, passing at the top to reverse polarity lavas. Preliminary K-Ar ages bracketed this sequence between 1018 ± 18 ka and 978 ± 17 ka. Therefore, the upper Jaramillo reversal at least appeared to be potentially recorded in this sequence. A more detailed paleomagnetic study was then carried out to more precisely delimit the reversal itself (see Laj et al., session EMRP3.4). We have undertaken 40Ar/39Ar incremental heating and unspiked K-Ar experiments on groundmass from four transitionally magnetized flows. The first transitional flow is K-Ar dated at 993 ± 18 ka and 40Ar/39Ar dated at 991 ± 13 ka, the second at 981 ± 17 ka (K-Ar) and 1000 ± 13 ka (40Ar/39Ar), the third at 950 ± 17 ka (K-Ar) and 1000 ± 8 ka (40Ar/39Ar) and the fourth at 984 ± 17 ka (K-Ar) and 977 ± 12 (40Ar/39Ar). 40Ar/39Ar ages and K-Ar ages (relative to FCT 28.02 Ma) are indistinguishable at 2σ. The age of the upper boundary of the Jaramillo event calculated combining 40Ar/39Ar ages and K-Ar ages is 992 ± 6 ka, in agreement with previous estimates.

  16. Photoionization of Ar VIII

    NASA Astrophysics Data System (ADS)

    Liang, Liang; Jiang, Wen-xian; Zhou, Chao

    2017-01-01

    The photoionization cross section, energy levels and widths of 22 Rydberg series (in the autoionization region) for Na-like Ar VIII were investigated by using of R-matrix method. The relativistic distorted-wave method is used to calculate the radial functions, and QB method of Quigly-Berrington [Quigley et al. 1998] is employed to calculate the resonance levels and widths. We have identified the formant in the figure of the photoionization cross section.

  17. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.

    PubMed

    Wu, J Y; Maniatis, T

    1993-12-17

    Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

  18. Primordial abundance of 40Ar

    NASA Astrophysics Data System (ADS)

    Sripada, V. S. Murty

    Primordial abundance of the isotope (40) Ar is still not known accurately. Recent results from Genesis could also not provide (40) Ar/ (36) Ar value of solar wind, due mainly to the overwhelming (40) Ar blank. A major part of (40) Ar is contributed by the radioactive decay of (40) K (half life = 1.25 Ga), even in the nebula, as the nebula grew old. Any attempt to determine this quantity needs a sample that satisfies the following criteria: A primitive mineral/phase that formed very early in the nebula, that can trap a large amount of noble gas (Ar); and a phase that acquires minimum amount (or total absence) of in situ produced components (cosmogenic and radiogenic) of Ar. Carbon phases in the ureilite meteorites and Phase Q from chondrites best fit this criteria. The minimum (40) Ar/ (36) Ar value so far observed in Phase Q is 0.2. Also, the relatively lower value of 1.035±±0.002 for trapped (129) Xe/ (132) Xe in ureilites, as compared to 1.042±±0.002 in Phase Q suggests that trapping of gases in ureilites might have predated that of Phase Q. If this interpretation is valid, ureilites are a better host of most primitive nebular Ar. Earlier attempts on ureilite studies in 1970s have yielded the lowest (40) Ar/ (36) Ar ratio in the meteorite Dayalpur, the major uncertainty for this value mostly coming from blank correction for (40) Ar/ (36) Ar. Recent developments in low blank extraction systems and more sensitive multi-collector noble gas mass spectrometers, as compared to 1970s have prompted us to make a fresh attempt in measuring this important quantity. We have analysed a number of ureilite acid residues by stepwise temperature extraction, using both pyrolysis and combustion techniques, for Ar to ascertain the trapped (40) Ar/ (36) Ar ratio in the solar nebula. These acid residues are mostly made of C rich phases, with only trace amounts of K (radiogenic parent of (40) Ar) and target elements for the production of cosmogenic Ar component. They mostly contain

  19. The incredible complexity of RNA splicing.

    PubMed

    Robert, Christelle; Watson, Mick

    2016-12-30

    Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets.

  20. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

    PubMed

    Treisman, R; Orkin, S H; Maniatis, T

    1983-04-14

    Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

  1. The Characterizations of Different Splicing Systems

    NASA Astrophysics Data System (ADS)

    Karimi, Fariba; Sarmin, Nor Haniza; Heng, Fong Wan

    The concept of splicing system was first introduced by Head in 1987 to model the biological process of DNA recombination mathematically. This model was made on the basis of formal language theory which is a branch of applied discrete mathematics and theoretical computer science. In fact, splicing system treats DNA molecule and the recombinant behavior by restriction enzymes and ligases in the form of words and splicing rules respectively. The notion of splicing systems was taken into account from different points of view by many mathematicians. Several modified definitions have been introduced by many researchers. In this paper, some properties of different kinds of splicing systems are presented and their relationships are investigated. Furthermore, these results are illustrated by some examples.

  2. Tumor microenvironment-associated modifications of alternative splicing.

    PubMed

    Brosseau, Jean-Philippe; Lucier, Jean-François; Nwilati, Hanad; Thibault, Philippe; Garneau, Daniel; Gendron, Daniel; Durand, Mathieu; Couture, Sonia; Lapointe, Elvy; Prinos, Panagiotis; Klinck, Roscoe; Perreault, Jean-Pierre; Chabot, Benoit; Abou-Elela, Sherif

    2014-02-01

    Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

  3. Practical reactor production of 41Ar from argon clathrate.

    PubMed

    Mercer, J R; Duke, M J; McQuarrie, S A

    2000-06-01

    The radionuclide 41Ar has many ideal properties as a gas flow tracer. However, the modest cross-section of 40Ar for thermal neutron activation makes preparation of suitable activities of 41Ar technically difficult particularly for low flux reactors. Argon can however be trapped in a molecular complex called a clathrate that can then be irradiated. We prepared argon clathrate and explored its irradiation and stability characteristics. Argon clathrate can be used to provide gigabecquerel quantities of 41Ar even with low power reactors.

  4. PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor.

    PubMed

    Maita, Hiroshi; Kitaura, Hirotake; Keen, T Jeffrey; Inglehearn, Chris F; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2004-11-01

    PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). However, RP9 is a rare disease and only two missense mutations have been described, so the report of a link between PAP-1 and RP9 was tentative. The precise cellular role of PAP-1 was also unknown at that time. We now report that PAP-1 localizes in nuclear speckles containing the splicing factor SC35 and interacts directly with another splicing factor, U2AF35. Furthermore, we used in vitro and in vivo splicing assays to show that PAP-1 has an activity, which alters the pattern of pre-mRNA splicing and that this activity is dependent on the phosphorylation state of PAP-1. We used the same splicing assay to examine the activities of two mutant forms of PAP-1 found in RP9 patients. The results showed that while one of the mutations, H137L, had no effect on splicing activity compared with that of wild-type PAP-1, the other, D170G, resulted in both a defect in splicing activity and a decreased proportion of phosphorylated PAP-1. The D170G mutation may therefore cause RP by altering splicing of retinal genes through a decrease in PAP-1 phosphorylation. These results demonstrate that PAP-1 has a role in pre-mRNA splicing and, given that three other splicing factors have been implicated in adRP, this finding provides compelling further evidence that PAP-1 is indeed the RP9 gene.

  5. ArsP: a methylarsenite efflux permease

    PubMed Central

    Chen, Jian; Madegowda, Mahendra; Bhattacharjee, Hiranmoy; Rosen, Barry P.

    2015-01-01

    Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals. PMID:26234817

  6. Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

    PubMed Central

    2010-01-01

    Background Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment. Results We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants. Conclusions TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of

  7. Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis

    SciTech Connect

    Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha; Conboy, John G.

    2009-02-03

    Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

  8. Splice Variants of the RTK Family: Their Role in Tumour Progression and Response to Targeted Therapy

    PubMed Central

    Abou-Fayçal, Cherine; Hatat, Anne-Sophie; Gazzeri, Sylvie; Eymin, Beatrice

    2017-01-01

    Receptor tyrosine kinases (RTKs) belong to a family of transmembrane receptors that display tyrosine kinase activity and trigger the activation of downstream signalling pathways mainly involved in cell proliferation and survival. RTK amplification or somatic mutations leading to their constitutive activation and oncogenic properties have been reported in various tumour types. Numerous RTK-targeted therapies have been developed to counteract this hyperactivation. Alternative splicing of pre-mRNA has recently emerged as an important contributor to cancer development and tumour maintenance. Interestingly, RTKs are alternatively spliced. However, the biological functions of RTK splice variants, as well as the upstream signals that control their expression in tumours, remain to be understood. More importantly, it remains to be determined whether, and how, these splicing events may affect the response of tumour cells to RTK-targeted therapies, and inversely, whether these therapies may impact these splicing events. In this review, we will discuss the role of alternative splicing of RTKs in tumour progression and response to therapies, with a special focus on two major RTKs that control proliferation, survival, and angiogenesis, namely, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-1 (VEGFR1). PMID:28208660

  9. STABILIZED1 Modulates Pre-mRNA Splicing for Thermotolerance1[OPEN

    PubMed Central

    Cho, Young-Hee

    2017-01-01

    High-temperature stress often leads to differential RNA splicing, thus accumulating different types and/or amounts of mature mRNAs in eukaryotic cells. However, regulatory mechanisms underlying plant precursor mRNA (pre-mRNA) splicing in the environmental stress conditions remain elusive. Herein, we describe that a U5-snRNP-interacting protein homolog STABILIZED1 (STA1) has pre-mRNA splicing activity for heat-inducible transcripts including HEAT STRESS TRANSCRIPTION FACTORs and various HEAT SHOCK PROTEINs for the establishment of heat stress tolerance in Arabidopsis (Arabidopsis thaliana). Our cell-based splicing reporter assay demonstrated STA1 acts on pre-mRNA splicing for specific subsets of stress-related genes. Cellular reconstitution of heat-inducible transcription cascades supported the view that STA1-dependent pre-mRNA splicing plays a role in DREB2A-dependent HSFA3 expression for heat-responsive gene expression. Further genetic analysis with a loss-of-function mutant sta1-1, STA1-expressing transgenic plants in Col background, and STA1-expressing transgenic plants in the sta1-1 background verified that STA1 is essential in expression of necessary genes including HSFA3 for two-step heat stress tolerance in plants. However, constitutive overexpression of the cDNA version of HSFA3 in the sta1-1 background is unable to execute plant heat stress tolerance in sta1-1. Consistently our global target analysis of STA1 showed that its splicing activity modulates a rather broad range of gene expression in response to heat treatment. The findings of this study reveal that heat-inducible STA1 activity for pre-mRNA splicing serves as a molecular regulatory mechanism underlying the plant stress tolerance to high-temperature stress. PMID:28223317

  10. Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing.

    PubMed

    Mayerle, Megan; Guthrie, Christine

    2016-05-01

    Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiaes how that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.

  11. An interspecific plant hybrid shows novel changes in parental splice forms of genes for splicing factors.

    PubMed

    Scascitelli, Moira; Cognet, Marie; Adams, Keith L

    2010-04-01

    Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization.

  12. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    PubMed

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program.

  13. Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.

    PubMed Central

    Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W

    1993-01-01

    The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs. Images PMID:8417357

  14. MAASE: An alternative splicing database designed for supporting splicing microarray applications

    PubMed Central

    ZHENG, CHRISTINA L.; KWON, YOUNG-SOO; LI, HAI-RI; ZHANG, KUI; COUTINHO-MANSFIELD, GABRIELA; YANG, CANZHU; NAIR, T. MURLIDHARAN; GRIBSKOV, MICHAEL; FU, XIANG-DONG

    2005-01-01

    Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication. PMID:16251387

  15. SAW: A Method to Identify Splicing Events from RNA-Seq Data Based on Splicing Fingerprints

    PubMed Central

    Ning, Kang; Fermin, Damian

    2010-01-01

    Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons. PMID:20706591

  16. Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells

    PubMed Central

    Bolton, Eric C.

    2015-01-01

    The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN