Harpold M M, Wilson M C, Darnell J E
Rockefeller University, New York, New York 10021.
Mol Cell Biol. 1981 Feb;1(2):188-98. doi: 10.1128/mcb.1.2.188-198.1981.
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.
我们通过使用一组先前构建的互补脱氧核糖核酸（DNA）克隆（Harpold等人，《细胞》17:1025 - 1035，1979年）作为特异性分子探针，在各种RNA:DNA杂交实验中，进一步分析了中国仓鼠卵巢（CHO）细胞胞质和核RNA中特定信使核糖核酸（mRNA）序列的代谢情况。与九个克隆中的每一个互补的大部分标记mRNA存在于多核糖体中，各个序列之间存在一些差异。在3小时或更短时间内标记的每种特异性mRNA的绝大多数存在于聚腺苷酸化[poly(A)+]部分。然而，在很长的标记时间后，每种序列在非聚腺苷酸化[poly(A)-]部分中的量增加，这表明poly(A)- RNA来源于poly(A)+ RNA。九个mRNA中的八个在胞质中的半衰期为8至14小时，而其中一个mRNA，即该组中最稀少的一个，半衰期约为3小时，略短一些。每种特异性长寿命mRNA在总标记mRNA中的比例随标记时间增加，这表明CHO细胞中最初标记的聚腺苷酸化[poly(A)+] mRNA的很大一部分，可能超过50%，半衰期小于3小时。对特定核序列和胞质序列标记动力学的定量分析表明，从包含这九个CHO序列的基因转录的mRNA序列中有很大一部分成功加工成了mRNA。然而，在核加工产生mRNA的过程中，其中两个CHO mRNA序列仅部分保守。这些研究表明，转录后两个水平的事件，即不同初级转录本的核加工效率差异和不同mRNA的胞质稳定性，可能参与了不同mRNA胞质浓度的决定。
