Effect of differential polyadenylation and cell growth phase on dihydrofolate reductase mRNA stability.
We have constructed tetracycline-responsive dhfr minigenes and transferred them to a Chinese hamster ovary cell DHFR-deficient deletion mutant to obtained cells in which dhfr transcription can be repressed by tetracycline (tet-off). DHFR mRNA half-life measured after the repression of transcription by tetracycline in these transfectants is about 1.5 h, which is significantly shorter than previously reported. In addition, we observed that DHFR mRNA is less stable in serum-starved cells than in exponentially growing cells. Given that the dhfr gene contains multiple polyadenylation sites, we analyzed the role of polyadenylation site usage on the stability of the resultant mRNA molecules. We found that DHFR mRNA is more stable when a strong polyadenylation site is used. Finally, we have observed that the relative lengths of the poly(A) tails for the different DHFR mRNA species correlated with their relative stability in growing versus resting cells.
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Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Ciudad
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Chasin
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D000818:Animals; D016466:CHO Cells; D002455:Cell Division; D006224:Cricetinae; D004794:Enzyme Repression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006207:Half-Life; D007700:Kinetics; D016296:Mutagenesis; D011061:Poly A / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D017384:Sequence Deletion; D013752:Tetracycline / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D012333:RNA, Messenger; D011061:Poly A; D013762:Tetrahydrofolate Dehydrogenase; D013752:Tetracycline