RRM
2008 Rustbelt RNA Meeting
RRM
|
|
|
|
|
Poster abstracts
Abstract:
Beta-thalassemia is an inherited disorder of hemoglobin production that results from the presence of a premature termination codon in the body of beta-globin mRNA. In murine erythroleukemia (MEL) cells, PTC-containing beta-globin mRNA is degraded by endonuclease cleavage and results in the accumulation of metastable mRNA decay intermediates. An earlier report suggested that these intermediates possess a cap-like modification at 5?f end [Lim, S.K., and Maquat, L.E. 1992. EMBO J. 11:3271-3278]. The first part of this study examined the cap status of these decay intermediates by their recovery with an immobilized monoclonal antibody to the trimethylcap or a cytoplasmic cap binding protein eIF4E. Both full-length mRNA and the smaller decay products were recovered, supporting the presence of a true cap. This was confirmed by cap analog competition for binding to eIF4E and susceptibility to hydrolysis by the cellular decapping enzyme Dcp2. Although mammalian capping enzyme (CE) is generally thought to be restricted to the nucleus, a population of cytoplasmic CE was identified by Western blotting, &alpha-32P GMP labeling of the active site intermediate and fluorescence microscopy. Cytoplasmic CE sediments at 140-200 kDa on glycerol gradients and immunoprecipitated cytoplasmic CE complex converts a 5?f-monophosphate (but not a 5?f-hydroxyl) RNA to 5?f GpppX. The recovery from stress was used to evaluate the biological function of cytoplasmic capping enzyme. Arsenite causes a generalized inhibition of translation and the ability of cells to recover from arsenite stress was reduced by expression of inactive capping enzyme in a form that is restricted to the cytoplasm. These data support a role for capping in the cytoplasm and suggest that some mRNAs may be stored in an uncapped state.
Keywords: endonuclease-mediated mRNA decay, cytoplasmic capping, cell stress
