Steady state and time course RNA blot analyses of PGK1 harboring endogenous À1 RF signals mRNA stability vectors were transformed into wild-type yeast (JD1158), upf1D or upf2D (JD1181 or JD1367), xrn1Á (JD1170), dcp1Á (JD1122), ski2Á (JD1345), ski3Á (JD19) and dom34Á (JD1363) cells. The EST2 mRNA stability vector (pJD754) was transformed into rpb1-1 (JD977) and rpb1-1/Upf À (JD978) cells and time courses were performed as described previously (26) . Total RNA was extracted with acid phenol/chloroform (pH = 4.5) from mid-logarithmic cell cultures (27) , or with Trizole ß Reagent following the manufacturer's directions (Invitrogen, Carlsbad, CA, USA). RNA (northern) blotting was performed as previously described (19) . Equal amounts of RNA (1, 2 or 4 mg) were separated through 1% agarose-formaldehyde gels. RNA samples were transferred and UV cross linked to Hybond-N-membranes (Amersham). Blots were hybridized with g[ 32 P] 5 0 -end-labeled oligonucleotides specific for U3 snoRNA (loading control) and the exogenous Renilla fragment (experimental). Messenger RNAs were identified using a GeneStorm phosphoimager (Bio-Rad) and quantified using QuantifyOne (Bio-Rad). Each experiment was repeated three or more times and averaged to generate graphs. Error bars for calculations including ratios of ratios may be approximated using either of the two following calculations. 'Average Ratio' is defined as the ratio of the two calculated values. Value ctrl is the value of the control (the denominator of the ratio) while Stdev ctrl is the calculated standard deviation of the control. Similarly, value exp and stdev exp are the value and standard deviations of the experimental (the numerator of the ratio). Rep ctrl and rep exp are the number of replicates performed for the control and experimental respectively. The error bars in the graphs of ratios of ratios use the approximated standard error.