RRM
2006
Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
PTC-containing human beta-globin mRNA is commonly used to study NMD in non-erythroid cells, where it decays in association with the nuclear fraction. In erythroid cells a PTC at codon 60/61 activates cytoplasmic endonuclease decay but has no impact on steady state mRNA in the nuclear fraction. We sought to determine the relationship between the cytoplasmic endonuclease-mediated decay of beta-globin mRNA in erythroid cells versus non-erythroid cells, and asked if the location of the PTC determines the sites of endonuclease cleavage. Beta-globin genes were generated with PTCs at codons 17, 22, 35, 39, 43, 60/61 and 90, and these were transiently transfected into murine erythroleukemia (MEL) cells. Steady-state levels of beta-globin mRNA were quantified in cytoplasmic and nuclear extracts by real-time PCR, and sites of endonuclease cleavage by S1 nuclease protection assay. In agreement with earlier work PTC-containing beta-globin mRNA is selectively lost from the cytoplasmic fraction of these cells, with the greatest impact on mRNAs with PTCs at codons 35, 39, 43, and 60/61. This was reversed by transfecting cells with dominant negative forms of Upf1, indicating cytoplasmic endonuclease decay constitutes a form of surveillance for beta-globin mRNA in erythroid cells. In addition we find that the location of the PTC has no impact on the position of endonuclease cleavage sites. When the same genes were analyzed in Cos-1 cells PTC-containing beta-globin mRNA is lost from the nuclear fraction, and with no evidence for production of endonuclease decay intermediates. These results demonstrate that different mechanisms are involved in the decay of PTC-containing beta-globin mRNA in erythroid versus non-erythroid cells.
Keywords: Nonsense-mediated decay, beta-globin, endonuclease
