mRNA lacking a 3′ polyA tail is not translated efficiently in wild-type eukaryotic cells, but is translated efficiently in yeast ski mutants. This enhanced expression could be due to altered translational specificity. However, as the SKI genes are required for 3′ mRNA degradation, it could be a consequence of inhibition of 3′ mRNA decay. Therefore, we asked if inhibition of 3′ decay of a polyA-minus mRNA in cis would allow its efficient expression in wild-type cells. Capped in vitro reporter transcripts were prepared with or without a 3′ cis-acting element known to inhibit 3′ degradation (oligoG) and electroporated into yeast cells. The addition of oligoG to a polyA-minus mRNA enhanced expression 30-fold in wild-type cells. This level of expression was the same as that for an oligoG-minus, polyA-minus transcript in a ski mutant. The addition of oligoG did not significantly enhance the expression of polyA-minus mRNA in a ski mutant. The oligoG-dependent increase in expression was due to an increase in initial rate of translation and an increase in the functional half-life of the mRNA, similar to the effects observed in a ski mutant. The enhanced expression of the oligoG-containing RNA did not require Pab1p. We conclude that the enhanced translation of polyA-minus RNA in a ski mutant is due to inhibition of 3′ mRNA degradation. Furthermore, a polyA-minus mRNA is expressed in wild-type cells when terminated in an element known to inhibit 3′ decay in cis.

A cell-free mRNA decay assay has been adapted to permit the kinetics of 3′ → 5′ exoribonuclease activities to be monitored in real time. RNA probes containing 5′ caps and 3′ poly(A) tails generated by transcription in vitro are 3′ labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3′ end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3′ untranslated region and an 85-base poly(A) tail. Following 3′ fluorescein labeling, the rate of 3′-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed.