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Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative
M M Harpold1, M C Wilson, J E Darnell
1Rockefeller University, New York, New York 10021.
Abstract:
We have further analyzed the metabolism of specific messenger ribonucleic acid (mRNA) sequences within the cytoplasmic and nuclear RNA of Chinese hamster ovary (CHO) cells by using a set of previously constructed complementary deoxyribonucleic acid (DNA) clones (Harpold et al., Cell 17:1025-1035, 1979) as specific molecular probes in a variety of RNA:DNA hybridization experiments. The majority of the labeled mRNA complementary to each of the nine clones was found in the polyribosomes, with some variation between individual sequences. The great majority of each specific mRNA labeled for 3 h or less was in the polyadenylated [poly(A)+] fraction. However, the amount of each sequence increased in the non-poly(A)+ [poly(A)-] fraction after very long label times, suggesting the derivation of the poly(A)- RNA from the poly(A)+ RNA. Eight of the nine mRNA's have cytoplasmic half-lives ranging from 8 to 14 h, whereas one of the mRNA's, the scarcest in the group, has a somewhat shorter half-life of approximately 3 h. The proportion of each of the specific long-lived mRNA's within the total labeled mRNA increased as a function of labeling time, indicating that a large fraction, probably greater than 50%, of the initially labeled poly(A)+ mRNA in CHO cells has a half-life of less than 3 h. A quantitative analysis of the kinetics of labeling of specific nuclear and cytoplasmic sequences indicated that a significant fraction of the mRNA sequences transcribed from genes containing these nine CHO sequences were successfully processed into mRNA. However, two of the CHO mRNA sequences were only partially conserved during nuclear processing to yield mRNA. These studies demonstrated that events at two post-transcriptional levels, differential nuclear processing efficiency of different primary transcripts and cytoplasmic stability of different mRNA's, can be involved in the determination of the cytoplasmic concentrations of different mRNA's.