Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU.

Abstract

In frog oocytes, certain maternal mRNAs receive poly(A) in the cytoplasm during progesterone-induced maturation. To analyze this reaction and to compare it to poly(A) addition in the nucleus, we injected short, synthetic RNA substrates into Xenopus oocytes. These RNAs contain only portions of the 3'-untranslated regions of appropriate mRNAs and end at the natural poly(A) site. We demonstrate that the nuclear and maturation-specific polyadenylation activities are distinct in substrate specificity and subcellular location. The sequence AAUAAA, contained in virtually all pre-mRNAs, is necessary for both activities. A second sequence element, UUUUUAU, activates poly(A) addition during maturation. UUUUUAU and AAUAAA are both necessary and virtually sufficient for maturation-specific polyadenylation: Poly(A) tails of between 50 and 300 nucleotides are added during maturation to RNAs containing both sequences but not to RNAs that lack either sequence. Before maturation, RNAs that contain AAUAAA are extended by just 10 nucleotides, presumably adenosines. The maturation-specific activity first appears within 1 hr of the time the nucleus breaks down but apparently does not require a nuclear component, as it is unaffected by enucleation. These observations, combined with those of others, lead us to speculate that polyadenylation may be responsible for the translational activation of a family of mRNAs essential for maturation.

Inactivation of protein kinase A is not required for c-mos translation during meiotic maturation of Xenopus oocytes

“…Capped, radiolabelled RNAs for polyadenylation assays were prepared by in vitro transcription of digested pcDNA3 177 bp c-mos using T7 RNA poly-merase in the presence of a 32 P-UTP as described (Fox et al, 1989).…”

Targeted translational regulation using the PUF protein family scaffold

“…Injected RNAs typically are stable, even if they lack poly(A), which simplifies analysis of translational activity. A variety of activation and repression domains have been identified in oocytes (9,10,26,28,33,36,37), and cytoplasmic poly(A) addition and removal events are well documented (30,(38)(39)(40)(41)(42)(43)(44).…”

Xenopus Cold-inducible RNA-binding Protein 2 Interacts with ElrA, the Xenopus Homolog of HuR, and Inhibits Deadenylation of Specific mRNAs

“…In general, polyadenylation of a cytoplasmic mRNA leads to its translational up-regulation, whereas deadenylation reduces translational activity. Several translationally repressed mRNAs in Xenopus oocytes that contain a specific cis-element, the cytoplasmic polyadenylation element (CPE), in their 3Ј-untranslated region (3Ј-UTR) have short poly(A) in the oocytes (6,7). Upon oocyte maturation, these mRNA receives elongated poly(A), which in turn results in translational activation.…”