CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

Abstract

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m 7 GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.Selective protein synthesis in oocytes, eggs, and early embryos of many organisms drives several critical aspects of early development, including meiotic maturation and entry into mitosis, establishment of embryonic axes, and cell fate determination. Protein synthesis is usually regulated at the initiation stage, mediated by the 5Ј m 7 GpppN mRNA cap structure bound by the translation initiation complex eIF4F, composed of eIF4E, the cap-binding protein, the RNA helicase eIF4A, and the large scaffold protein eIF4G, which has a consensus binding site YXXXXL for eIF4E, and additional sites for eIF3 and the poly(A)-binding protein. eIF3 recruits the small ribosomal subunit, whereas the eIF4E-eIF4G-poly(A)-binding protein relay results in the so-called "closed loop" model, responsible for the synergistic enhancement of translation by capped and polyadenylated mRNAs. A hallmark of translational control mechanisms is the role of mRNA-binding proteins, which recognize specific (and usually) 3Ј-UTR 2 cis-elements and influence the recruitment of the small ribosomal subunit to the 5Ј cap (reviewed in Refs. 1-3). Probably, the best studied mRNA-binding protein is CPEB1 (cytoplasmic polyadenylation-binding protein 1), characterized in flies, worms, clams, Aplysia, Xenopus, and mammals, which in its conserved C terminus contains two tandem RNA reco...

Nucleocytoplasmic Traffic of CPEB1 and Accumulation in Crm1 Nucleolar Bodies

“…Although CPEB1 was nearly undetectable in the nuclear fraction of untreated cells, it accumulated in the nucleus after LMB treatment ( Figure 2B), confirming that the endogeneous protein enters the nucleus, and is exported via the CRM1 machinery. As a control, we analyzed the Rck/p54 DEAD box helicase, which belongs to the same cytoplasmic ribonucleoprotein complex as CPEB1 in oocytes (Minshall et al, 2007). In concordance with previous reports (Smillie and Sommerville, 2002), Rck/p54 was cytoplasmic and not sensitive to LMB treatment.…”

“…Beside mRNA, the cytoplasmic CPEB1 complex contains a set of proteins, including the DEAD-box helicase Rck/p54, which is required for mRNA repression (Minshall et al, 2007). To assess its presence in nuclear CPEB1 foci, cells were transfected with CPEB1-GFP and 24 h later, were immunostained with anti-p54 antibody.…”

“…The domains responsible for RNA binding are contained in its C-terminal moiety, which contains two RNA recognition motifs (RRMs) and a zinc-finger domain. CPEB represses translation by binding to eIF4E-T (Minshall et al, 2007) and maskin (Stebbins-Boaz et al, 1999) in the early and late oocyte, respectively. In turn, these proteins bind and block the translation initiation factor eIF4E on the capped extremity of the mRNA.…”

The SESA network links duplication of the yeast centrosome with the protein translation machinery

“…Thus, in respect of specificity SESA regulation is similar to the translation control of mRNAs containing a cytoplasmic polyadenylation element (CPE) by Maskin or 4E-T (4E-BP) in Xenopus laevis. In this case the CPE-binding protein (CPEB) acts as the specific RNAbinding protein (Stebbins-Boaz et al 1999;Minshall et al 2007). Another such example is the translational repression of oskar mRNA in Drosophila by Cup (a 4E-BP) where Bruno acts as the mRNA-binding protein (Nakamura et al 2004;Chekulaeva et al 2006; for reviews, see Richter and Sonenberg 2005;Sonenberg and Hinnebusch 2009).…”

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…In Xenopus spp. oocytes, the eIF4E1b protein was found to bind eIF4E transporter and cytoplasmic polyadenylation element binding protein to form a translation-repressing complex (Minshall et al, 2007). Drosophila species have undergone extensive expansion of EIF4E-encoding loci to as many as seven different Class I eIF4E isoforms (Tettweiler et al, 2012).…”