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. 2000 Mar;6(3):458–464. doi: 10.1017/s1355838200991970

A fluorescence-based assay for 3' --> 5' exoribonucleases: potential applications to the study of mRNA decay.

G M Wilson 1, H Lu 1, Y Sun 1, A Kennedy 1, G Brewer 1
PMCID: PMC1369927  PMID: 10744029

Abstract

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.

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Selected References

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