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1.
Nat Commun ; 9(1): 3112, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082712

RESUMO

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.


Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Deleção de Genes , Inativação Gênica , Engenharia Genética , Genoma Humano , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Transplante de Neoplasias , Antígeno Neuro-Oncológico Ventral , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Telomerase/metabolismo , Telômero/ultraestrutura
2.
J Biol Chem ; 283(45): 30585-95, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18782762

RESUMO

Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: . Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins.


Assuntos
Clostridium perfringens/metabolismo , Endotoxinas/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Receptores Imunológicos/metabolismo , Motivos de Aminoácidos/genética , Animais , Células CHO , Claudina-4 , Clostridium perfringens/química , Cricetinae , Cricetulus , Endotoxinas/química , Endotoxinas/genética , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície/métodos
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