| During the life span of a cell, frameshift and nonsense mutations can arise that could lead to the production deleterious truncated proteins. One mechanism developed to eliminate nonsense-containing mRNAs is nonsense-mediated mRNA decay (NMD).; Because many nonsense-containing mRNAs in mammals are degraded while associated with the nucleus, I examined the localization of wild type and nonsense-containing RNAs by fluorescence in situ hybridization (FISH). Unexpectedly, nonsense-containing RNAs were present in larger and brighter transcription spots than wild type RNAs and steady-state pre-mRNA levels were also elevated. Furthermore, this increase was dependent upon the truncated open reading frame.; To further examine the nonsense-containing transcription spots, I manipulated HeLa cells containing wild type or mutant stable transgenes using translation inhibitors, known to abrogate both translation and NMD, and depletion by RNAi of known and putative NMD factors. Interestingly, inhibitors that stalled the ribosome caused an increase in signal for the mutant RNAs while an inhibitor that released the ribosomes and RNAs caused a decrease in signal at the mutant transcription spots, indicating the differential effects observed are not due to loss of rapidly degraded factor. Depletion of hUpf1p, a known NMD factor, or eIF4AIII, a nuclear translation initiation factor homolog, inhibited NMD, but had different effects on the transcription spots. In hUpf1-depleted cells, the transcription spots of wild type and mutant RNAs were unaffected or slightly brighter. In eIF4AIII-depleted cells, however, transcription spots of both genes were dimmer. Finally, loss of Rrp6/PM/Sc1–100, an exosome component had no effect on NMD although both transcription spots were dimmer. Finally, I localized ribosomal proteins in multiple cells types to see if nuclear translation could be responsible for reading frame recognition. Although the proteins were nuclear, immunofluorescence does not allow determination of whether the proteins are part of active ribosomes. Although I was unable to account for why nonsense-containing pre-mRNAs accumulated, further work with the inhibitors and protein depletions may elucidate the mechanism. |
