No abstract

In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5-to-3 exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5 and 3 ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.Messenger RNA degradation contributes significantly to the regulation of gene expression. In eukaryotes, elucidation of a number of mRNA degradation pathways is under way (for reviews, see refs 1-4). Presently, these pathways are better understood in yeast than in mammalian cells. Both in yeast and in mammalian cells, degradation of most polyadenylylated mRNAs appears to be initiated by poly(A) shortening: transcriptional pulse-chase experiments have shown that shortening of the poly(A) tail precedes mRNA degradation (5, 6). Some regulatory sequence determinants affect mRNA stability by modulating the rate of deadenylylation, whereas others modulate a later degradation step (5-7). This later step has been elucidated in yeast: deadenylylation at the 3Ј end triggers mRNA decapping at the 5Ј end, which is then followed by 5Ј-to-3Ј exonucleolysis (refs. 8 and 9 and references therein). In some specific circumstances, other degradation pathways are observed: exonucleolysis from the 3Ј end can occur when the 5Ј-to-3Ј exonuclease is inactive, and a decapping pathway independent of poly(A) tail shortening is involved in the degradation of mRNAs that show premature translation termination (9-11).In mammalian cells, the degradation pathway that follows deadenylylation is not well understood. Uncapped mRNAs are less stable than their capped counterparts in cell extracts, and enzymatic activities that catalyze mRNA decapping and 5Ј-to-3Ј exonucleolysis have been identified (refs. 1 and 3 and references therein). Furthermore, there is a conservation between yeast and mammalian cells of a functional interaction between the 5Ј and 3Ј ends of mRNAs: in both systems, these two ends contribute to translational control (e.g., see refs. 12 and 13). It is thus tempting to speculate that deadenylylation triggers a decapping-dependent degradation pathway in mammalian cells as well. However, a direct demonstration is lacking.We have developed a revers...

We report gene transfer to the Edinburgh insertional mutant mouse (cf/cf), delivering CFTR cDNA-liposome complexes into the airways by nebulization. We show full restoration of cAMP related chloride responses in some animals and demonstrate, in the same tissues, human CFTR cDNA expression. Overall, a range of correction was seen with restoration of about 50% of the deficit between wild type mice and untreated cf/cf controls. We report modest correction in the intestinal tract following direct instillation and provide initial encouraging safety data for both the respiratory and intestinal tract following the liposome mediated gene delivery. The non-viral nature and potentially lower immunogenicity of DNA-liposomes suggest that this may offer a therapeutic alternative to adenoviral therapies.