The mechanism and regulation of deadenylation: identification and characterization of Xenopus PARN.
In Xenopus oocytes, the deadenylation of a specific class of maternal mRNAs results in their translational repression. Here we report the purification, characterization, and molecular cloning of the Xenopus poly(A) ribonuclease (xPARN). xPARN copurifies with two polypeptides of 62 kDa and 74 kDa, and we provide evidence that the 62-kDa protein is a proteolytic product of the 74-kDa protein. We have isolated the full-length xPARN cDNA, which contains the tripartite exonuclease domain conserved among RNase D family members, a putative RNA recognition motif, and a domain found in minichromosome maintenance proteins. Characterization of the xPARN enzyme shows that it is a poly(A)-specific 3' exonuclease but does not require an A residue at the 3' end. However, the addition of 25 nonadenylate residues at the 3' terminus, or a 3' terminal phosphate is inhibitory. Western analysis shows that xPARN is expressed throughout early development, suggesting that it may participate in the translational silencing and destabilization of maternal mRNAs during both oocyte maturation and embryogenesis. In addition, microinjection experiments demonstrate that xPARN can be activated in the oocyte nucleus in the absence of cytoplasmic components and that nuclear export of deadenylated RNA is impeded. Based on the poly(A) binding activity of xPARN in the absence of catalysis, a model for substrate specificity is proposed.
Copeland
P R
PR
Department of Biology, University of Virginia, Charlottesville 22903, USA. copelap@ccf.org
Wormington
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014981:Xenopus
D018076:DNA, Complementary; D005095:Exoribonucleases; D011061:Poly A