Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3Ј-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3Ј-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturationspecific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

No abstract

is also initiated by deadenylation (Wilson and Treisman, D-35392 Giessen, Germany and 3 Department of Biology, University of 1988; Shyu et al., 1991; Chen and Shyu, 1995 1996). During oocyte maturation, deadenylation does not are not detectable in the enzyme preparation, and require specific cis-elements and is a default pathway PARN itself binds to m 7 GTP-Sepharose and is eluted for mRNAs that do not undergo compensatory poly(A) specifically with the cap analog m 7 GTP. Xenopus PARN elongation (Fox and Wickens, 1990; Varnum and is known to catalyze mRNA deadenylation during Wormington, 1990). In contrast, certain mRNAs which oocyte maturation. The enzyme is depleted from oocyte are polyadenylated during meiotic maturation contain extract with m 7 GTP-Sepharose, can be photocross-3Ј-UTR elements that promote their subsequent deadenyllinked to the m 7 GpppG cap and deadenylates ation after fertilization. Thus, the translation of these m 7 GpppG-capped RNAs more efficiently than ApppGmRNAs is restricted to mature oocytes (Bouvet et al., capped RNAs both in vitro and in vivo. These data 1994; Legagneux et al., 1995). In both cases, deadenylation provide additional evidence that PARN is responsible does not destabilize mRNAs immediately, but is a prefor deadenylation during oocyte maturation and sugrequisite for their degradation at later stages of developgest that interactions between 5Ј cap and 3Ј poly(A) ment (Audic et al., 1997; Gillian-Daniel et al., 1998; tail may integrate translational efficiency with mRNA Voeltz and Steitz, 1998). The uncoupling of deadenylation stability.from mRNA decay in gametes and embryos contrasts with Keywords: cap structure/deadenylation/mRNA stability/ both yeast and metazoan cells in which poly(A) removal oocyte maturation/poly(A) tails rapidly promotes mRNA degradation. Both the dependence of decapping on prior deadenylation and the inhibition of translation initiation by poly(A)