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Sainte-Foy-lès-Lyon, France

Ash P.E.,Mayo Medical School | Zhang Y.-J.,Mayo Medical School | Roberts C.M.,University of Colorado at Boulder | Saldi T.,University of Colorado at Boulder | And 4 more authors.
Human Molecular Genetics | Year: 2010

RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein. © The Author 2010. Published by Oxford University Press. All rights reserved. Source

Anantharaman D.,International Agency for Research on Cancer IARC | Gheit T.,Infections and Cancer Biology Group | Waterboer T.,International Agency for Research on Cancer | Waterboer T.,German Cancer Center | And 27 more authors.
Journal of the National Cancer Institute | Year: 2013

Background Human papillomavirus (HPV) is causally implicated in a subset of cancers of the upper aero-digestive tract (UADT).MethodsAssociations between type-specific HPV antibodies were examined among 1496 UADT cancer case subjects and 1425 control subjects by estimating odds ratios (ORs) in logistic regression analyses adjusted for potential confounders. The agreement between serology and tumor markers of HPV infection, including presence of HPV DNA and p16 expression, were examined in a subset of tumors.ResultsHPV16 L1 seropositivity was associated with increased risk of oral cavity and oropharyngeal cancer (OR = 1.94, 95% confidence interval [CI] = 1.03 to 3.65; OR = 8.60, 95% CI = 5.21 to 14.20, respectively). HPV16 E6 antibodies were present in 30.2% of oropharyngeal case subjects and only 0.8% of control subjects (OR = 132.0, 95% CI = 65.29 to 266.86). Combined seropositivity to HPV16 E6 and E7 was rare (n = 1 of 1425 control subjects). An agreement of 67% was observed between HPV16 E6 serology and the corresponding presence of an HPV-related cancer: four of six HPV DNA-positive/p16-overexpressing tumors were HPV16 E6 antibody positive. An HPV16 independent association was observed for HPV18 and oropharyngeal cancer (OR = 8.14, 95% CI = 2.21 to 29.99 for HPV18 E6 seropositivity) and HPV6 and laryngeal cancer (OR = 3.25, 95% CI = 1.46 to 7.24 for HPV6 E7 seropositivity). ConclusionsThese results confirm an important role for HPV16 infection in oropharyngeal cancer. HPV16 E6 antibodies are strongly associated with HPV16-related oropharyngeal cancers. Continuing efforts are needed to consider both HPV serology and p16 staining as biomarkers relevant to the etiology and natural history of HPV16-related oropharyngeal tumors. These results also support a marginal role for HPV18 in oropharyngeal cancer and HPV6 in laryngeal cancer. © 2013 The Author. Source

Falanga A.,Molecular Pathology Group | Stojanovic O.,University of Zagreb | Kiffer-Moreira T.,Sanford Burnham Institute for Medical Research | Pinto S.,University of Zagreb | And 4 more authors.
Nucleic Acids Research | Year: 2014

Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function. © 2014 The Author(s) 2014. Source

Dhir A.,Molecular Pathology Group | Buratti E.,Molecular Pathology Group | Van Santen M.A.,Max Planck Institute for Biophysical Chemistry | Luhrmann R.,Max Planck Institute for Biophysical Chemistry | Baralle F.E.,Molecular Pathology Group
EMBO Journal | Year: 2010

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. © 2010 European Molecular Biology Organization. Source

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