Determination of mRNA half-life in HeLa cultures by a poly(A)-independent direct analysis of specific radioactivity of mRNA.
mRNA specific radioactivity in HeLa cultures exposed to (3H)uridine (10 muM) was determined directly by a highly selective poly(A)-independent method which we have described previously. Neither uridine in mRNA nor UTP approached the specific radioactivity of the exogenous (3H)uridine, but attained steady-state specific radioactivities which remained a third below the value of the added precursor. Using the labeling data for the evaluation of mRNA turnover, previously described by Greenberg, mRNA half-life in exponentially growing HeLa cultures was found to be 0.87 times the cell doubling time. Decay curves of mRNA in prelabeled cultures were in accordance with these values (half-life equals 0.79 times the cell doubling time) when corrected for growth and also for "reutilization" which was accomplished by relating uridine labeling in mRNA to UTP specific radioactivity. The experiments showed that an exact evaluation of mRNA turnover is possible only when the following points are taken into account. a) A constant supply of exogenous labeled uridine must be provided to guarantee a constant specific radioactivity of UTP. b) Labeling of CTP and of cytidine in RNA are delayed when compared with UTP and uridine in RNA. Corrections for cytidine labeling in RNA are therefore required. c) As rRNA approached a definitely lower steady-state specific radioactivity than mRNA, mRNA specific radioactivities must be determined directly (i.e. by radioactivity and absorbance at 260 nm in isolated mRNA fractions) in order to evaluate true turnover of this RNA species.
D002452:Cell Count; D002499:Centrifugation, Density Gradient; D003597:Cytosine Nucleotides / Q000378:metabolism; D006207:Half-Life; D006367:HeLa Cells / Q000378:metabolism*; D007700:Kinetics; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism*; D014316:Tritium; D014500:Uracil Nucleotides; D014529:Uridine / Q000378:metabolism
D003597:Cytosine Nucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014500:Uracil Nucleotides; D014316:Tritium; D012313:RNA; D014529:Uridine