RRM
2007 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
β-thalassemia is an inherited disorder of hemoglobin production that results from the presence of a premature termination codon in the body of β-globin mRNA. In murine erythroleukemia (MEL) cells, PTC-containing β-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'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, susceptibility to hydrolysis by tobacco acid pyrophosphatase, and removal of the cap with Dcp2. Mammalian capping enzyme (MCE) is generally thought to be restricted to the nucleus. Using a sensitive guanylylation assay, a cytoplasmic MCE-containing complex was identified that sediments at 140-200 kDa on glycerol gradients. Capping enzyme transfers a GMP to the 5'-diphosphate end of RNA via a covalent enzyme-GMP intermediate to generate the cap structure. However, the endonuclease that cleaves β-globin mRNA generates cleavage products with a '-monophosphate end. We present evidence for a heretofore unseen 5'-monophosphate kinase recovered with epitope-tagged capping enzyme from mammalian cells that facilitates addition of GMP onto RNA with a 5' monophosphate but not onto RNA lacking 5' phosphate. These results identify a potential new role for capping enzyme in modifying endonuclease-generated decay products, perhaps for subsequent decay.
Keywords: NMD, decay intermediates, capping enzyme
