A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors.
Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNA's in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges.
Harrold
S
S
University of Pittsburgh School of Medicine, Department of Molecular Genetics and Biochemistry, PA.
Genovese
C
C
Kobrin
B
B
Morrison
S L
SL
Milcarek
C
C
D000818:Animals; D016923:Cell Death; D002461:Cell Line, Transformed / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D006207:Half-Life; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics; D051379:Mice; D009101:Multiple Myeloma / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D012332:RNA, Heterogeneous Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007136:Immunoglobulins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D003609:Dactinomycin; D011061:Poly A; D004004:Dichlororibofuranosylbenzimidazole