Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

Contemporaneous isolation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and poly(A) polymerase from rat liver mitochondria.

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation.     

A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines

The rate of mRNA turnover is an important determinant of levels of gene expression. Although this process has been studied extensively in mammalian cells and yeast, relatively little is known about the mRNA decay pathways in insects. Our analysis found that the vast majority of components of the mRNA decay machinery are conserved between humans and mosquitoes. Moreover, the half-lives of Aedes albopictus mRNAs are within a similar range to those of mammalian mRNAs. In order to investigate mechanistic aspects of mRNA decay in mosquitoes, we developed an in vitro system using cytoplasmic S100 extracts from A. albopictus C6/36 cells. Using this decay assay, we show here that all the pathways of mRNA turnover that have been observed in mammalian cells (deadenylation, decapping, 3′-to-5′ exonucleolytic decay and 5′-to-3′ exonucleolytic decay) are active in C6/36 extracts. Finally, we present compelling evidence that the major deadenylase in C6/36 extracts is likely to be a homolog of the human poly(A) specific ribonuclease, PARN. Our results suggest a high level of conservation in the factors and pathways of mRNA decay between mosquitoes and humans.