Nonsense-mediated mRNA decay of the human HEXA mRNA

Many eukaryotic mRNAs harboring an early translation termination codon have been shown to undergo rapid degradation by nonsense-mediated mRNA decay (NMD). This is a process by which cells recognize and degrade nonsense-containing mRNAs to prevent possibly toxic effects of truncated proteins. The mechanism of NMD in mammalian cells is not well understood and the cellular site remains a debatable issue. Most mammalian nonsense mRNAs are degraded in or associated with the nucleus, though a few are degraded in the cytoplasm. Ongoing translation and at least one downstream intron are considered essential to NMD.;I have examined the NMD of a mutant HEXA message in cells derived from a Tay-Sachs disease patient homozygous for the common exon 11 frameshift mutation 1278ins4. The level of the mutant mRNA was nearly undetectable in lymphoblasts and the translational inhibitor cycloheximide increased the nonsense HEXA message level to 40% of normal. Once translation resumed after removal of cycloheximide, the nonsense HEXA mRNA rapidly decreased to its low level, showing that ongoing translation was a necessary process for the decay of this transcript.;I found the cycloheximide-stabilized nonsense message to be elevated in the cytoplasmic fraction, and fractionation on a sucrose gradient showed it to be associated with polysomes. Five hours after release from cycloheximide, the nonsense mRNA disappeared, while the normal message continued to associate with polysomes. This showed that the cytoplasmic pool of nonsense HEXA message was sensitive to NMD, unlike most other mammalian transcripts.;I then used a cell culture transfection system in which various HEXA minigenes were expressed in Chinese hamster ovary cells to determine the role of introns in the degradation of nonsense HEXA mRNA. Analysis showed that contrary to findings in other systems, introns were not absolutely required to promote the decay of the nonsense HEXA message; however, their presence enhanced the decrease in message level. My data, together with the published results of others, suggest that nonsense mediated mRNA decay in mammalian cells may be a complex multi-pathway phenomenon, with different transcripts following different pathways.