MOLECULAR REPRODUCTION AND DEVELOPMENT 75:1716–1725 (2008) Regulation of Cap-Dependent Translation Initiation in the Early Stage Porcine Parthenotes ANDREJ S ˇ US ˇ OR, 1 LUCIE JELI ´ NKOVA ´ , 1 PAVLA KARABI ´ NOVA ´ , 1 HELMUT TORNER, 2 WOLFGANG TOMEK, 2 HANA KOVA ´ R ˇ OVA ´ , 1 AND MICHAL KUBELKA 1 * 1 Institute of Animal Physiology and Genetics, Libechov, Czech Republic 2 Research Institute for the Biology of Farm Animals, Dummerstorf-Rostock, Germany ABSTRACT The binding of mRNAs to ribo- somes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5 0 -cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and asso- ciation with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regu- lator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphory- lation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9–15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes de- creases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development. Mol. Reprod. Dev. 75: 1716–1725, 2008. ß 2008 Wiley-Liss, Inc. Key Words: meiosis; translation initiation; porcine parthenotes; zygote development INTRODUCTION The regulated translation of mRNAs influences a large number of biological processes, including the cell cycle (Mendez and Richter, 2001; Pyronnet and Sonenberg, 2001; Groisman et al., 2002), growth, embryogenesis (Riechmann and Ephrussi, 2001; Niess- ing et al., 2002), and germ line development (Morris, 1995; Pain, 1996; Hake and Richter, 1997; Clemens and Bommer, 1999; Saffman and Lasko, 1999). In eukaryotes, the rate of cap-dependent protein synthesis is mainly determined by the evolutionary conserved eukaryotic translation initiation factor 4F (eIF4F), which is involved in the regulation of mRNA binding to ribosomes in conjunction with eIF4B (eukaryotic trans- lation initiation factor 4B). EIF4F is a complex, which is composed of three subunits termed eIF4E, eIF4A, and eIF4G (Shatkin, 1976; Gingras et al., 1999). 4E binds to the 7-methyl-GTP (m 7 GpppN) cap structure at the 5 0 -end of the mRNA, while eIF4A is an RNA- dependent ATPase that is thought to unwind the secondary structure present in the 5 0 -untranslated region of mRNAs. The role of eIF4G is well known in the initial formation of the eIF4F and the formation of the pre-initiation complex at the mRNA cap site. It may have an additional role in the final formation of the initiation complex at the start codon (Gebauer and Hentze, 2004). EIF4G also interacts with the MAP kinase-activated protein kinase (Mnk1) and the poly(A)- binding protein (PABP). Mnk1 phosphorylates and activates eIF4E via binding to eIF4G (Pyronnet et al., 1999). The interaction of eIF4F with repressed mRNA is necessary for the interaction of the ribosomal 40S subunit with the 5 0 -end of mRNA and for efficient initiation of translation (Sonenberg, 1988). The activity of most of the factors that participate in mRNA-ribosome binding (including eIF4B, eIF4E and eIF4G) is regulated mostly by phosphorylation (Pain, 1996; Morley, 1997). From those, the best studied so far is eIF4E, the cap-binding protein subunit of eIF4F complex. A number of studies have addressed the question of importance of eIF4E phosphorylation for the cap-dependent translation. EIF4E phosphorylated ß 2008 WILEY-LISS, INC. Grant sponsor: Grant Agency of the Czech Republic; Grant numbers: 524/04/0104, 524/07/1087; Grant sponsor: Deutsche Forschungsge- meinschaft; Grant number: 178 2-3. *Correspondence to: Dr. Michal Kubelka, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic. E-mail: kubelka@iapg.cas.cz Received 7 December 2007; Accepted 20 February 2008 Published online 2 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20913