RESUMEN
The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.
Asunto(s)
Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Procesamiento Postranscripcional del ARN , Humanos , Regiones no Traducidas 5' , ADN Helicasas/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVES: Asbestos is the name given to a group of naturally occurring silicate mineral fibres that were widely used in industry during the 20th century due to their desirable physical properties. Although use in the USA has fallen over the last three decades, significant exposure in the developing world continues and the burden of disease is considerable. Asbestos is a known risk factor for several malignant diseases, including lung cancer and mesothelioma, and has more recently been implicated in pharyngeal and laryngeal cancer. However, studies of asbestos and cancers of the larynx or pharynx with adequate sample size that control for major head and neck squamous cell carcinoma (HNSCC) risk factors remain relatively sparse. METHODS: We report findings from a case-control study of 674 incident male HNSCC cases from the greater Boston region and 857 population-based male controls, matched on age (±3 years), sex, and town or neighbourhood of residence. Multivariable logistic regression was used to assess the association between occupational asbestos exposure and HNSCC by primary tumour site. RESULTS: 190 cases (28.2%) and 203 controls (23.7%) reported occupational exposure to asbestos. Occupational asbestos exposure was associated with elevated risk of pharyngeal carcinoma in men (OR 1.41, 95% CI 1.01 to 1.97), adjusted for age, race, smoking, alcohol consumption, education, income and HPV16 serology, with borderline increasing risk for each decade in the exposed occupation (OR 1.10, 95% CI 0.99 to 1.23). CONCLUSIONS: These observations are consistent with mounting evidence that asbestos is a risk factor for pharyngeal cancer.