A fluorescence-based assay for 3' --> 5' exoribonucleases: potential applications to the study of mRNA decay.
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.
Wilson
G M
GM
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Piscataway 08854, USA.
Lu
H
H
Sun
Y
Y
Kennedy
A
A
Brewer
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002474:Cell-Free System; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D020014:K562 Cells; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D002457:Cell Extracts; D012333:RNA, Messenger; D011061:Poly A; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D019793:Fluorescein