Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.

Abstract

Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from -90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.

RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2

“…Because the 3′ variability in the CPE motif composition and length (45)(46)(47)(48) can obscure the de novo analysis, we searched both datasets for CPE variants containing either A or G in fifth position (Fig. 3B) and calculated enrichment over background 3′ UTR control.…”

The mechanism and regulation of deadenylation: Identification and characterization of Xenopus PARN

“…The role of poly(A) as a determinant of maternal mRNA translation during the meiotic maturation or subsequent fertilization of Xenopus oocytes is well established (Richter, 2000)+ This regulatory system discriminates between classes of mRNAs that are either polyadenylated or deadenylated during maturation+ One class of mRNAs, exemplified by G10, c-mos, and B4, contains the 39 UTR localized cis-sequences required for polyadenylation and subsequent translational activation (Dworkin & Dworkin, 1985;Fox et al+, 1989;McGrew et al+, 1989;Wormington, 1994)+ The cis sequences required for polyadenylation are the U-rich cytoplasmic polyadenylation element (CPE) and the ubiquitous nuclear polyadenylation element (AAUAAA) (McGrew & Richter, 1990;Paris & Richter, 1990)+ The deletion or mutational inactivation of either of these elements prevents both polyadenylation and translation (Fox et al+, 1989;McGrew et al+, 1989)+ In contrast, poly(A) removal is a default reaction deadenylating messages that lack a CPE such as those encoding ribosomal proteins and actin (Fox & Wickens, 1990;Varnum & Wormington, 1990)+ Deadenylated messages are dissociated from polysomes, thus preventing further translation+ Although the poly(A) tail is not necessarily sufficient for translatability (McGrew et al+, 1989), in no case has deadenylation been uncoupled from translational inactivation+ For example, the overexpression of poly(A)-binding protein (PABP) in Xenopus oocytes inhibits both maturation-specific deadenylation and translational silencing (Wormington et al+, 1996)+ The activity responsible for deadenylation in mature oocytes is initially nuclear associated, as poly(A) removal is a late maturation event that cannot be detected prior to nuclear envelope breakdown and is prevented if oocytes are enucleated prior to maturation (Varnum et al+, 1992)+ Importantly, Xenopus is the only system for which an in vivo function for deadenylation has been described (Fox & Wickens, 1990;Varnum & Wormington, 1990)+ The role of poly(A) in translation has been under intense scrutiny over the past few years (Sachs et al+, 1997;Sachs & Varani, 2000)+ The closed loop model of mRNA translation originally proposed by Munroe and Jacobson (1990) has been validated by subsequent biochemical and genetic evidence of interactions between the PABP and the cap-binding complex in yeast and mammalian systems (Tarun & Sachs, 1996;…”

The DAZL and PABP families: RNA-binding proteins with interrelated roles in translational control in oocytes

“…CPEs are the best characterized and have been predominantly studied in X. laevis oocytes (McGrew et al 1989). CPEs function in collaboration with the hexanucleotide (hex) signal AAUAAA (see (Mandel et al 2008) for review of nuclear polyadenylation), and both elements are binding sites for multiprotein complexes that can interact and are reviewed in detail elsewhere (Radford et al 2008).…”