Methods for measuring tissue RNA turnover.

Abstract

Measuring RNA turnover is important because of the significance of rRNA, tRNA and mRNA in tissue protein synthesis. Changes in turnover of each of these species precede important cellular events such as hormone or cytokine action or cell-division itself. Isotopic methods have relied on decay of pulse-labelled RNA or on incorporation of isotopically-labelled precursors. However, recycling of labels may lead to under or overestimation of synthesis rates respectively. The labelling of the intracellular precursor pool must be known if accurate RNA synthesis rates are to be calculated from the degree of incorporation. However, the intracellular nucleotide pools may be anatomically or metabolically compartmented (i.e. via de novo or salvage synthesis routes) and this complicates many study designs. The use of[methyl-14C]- or [methyl-3H]methionine as a means of labelling methylated nucleosides in RNA and protein simultaneously is described in addition to new stable isotopic techniques based on 13C-glycine as a de novo label. Urinary excretion of the numerous modified nucleosides in cellular RNA can be used to calculate whole-body turnover rates of each of the major RNA species. Examples of the effects of critical-illness and glutamine supplementation on RNA turnover are given. We conclude by suggesting that whole-body RNA turnover rates have been significantly underestimated and that this has implications for nutritional therapy, especially with regard to nucleotide supplementation.