A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease.
We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m(7)G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure and poly(A) degradation at the 3' end of the RNA.
Martinez
J
J
Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden.
Ren
Y G
YG
Thuresson
A C
AC
Hellman
U
U
Astrom
J
J
Virtanen
A
A
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D005095:Exoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D009068:Movement; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012315:RNA Caps / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D000249:Adenosine Monophosphate; D005095:Exoribonucleases; D010446:Peptide Fragments; D012315:RNA Caps