Greco Hernández

Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families

Protein synthesis in eukaryotic cells is fundamental for gene expression. This process involves the binding of an mRNA molecule to the small ribosomal subunit in a group of reactions catalyzed by eukaryotic translation initiation factors (eIF) eIF4. To date, the role of each of the four eIF4, i.e. eIF4E, eIF4G, eIF4A and eIF4B, is well established. However, with the advent of genome-wide sequencing projects of various organisms, families of genes for each translation initiation factor have been identified. Intriguingly, recent studies have now established that certain eIF4 proteins can promote or inhibit translation of specific mRNAs, and also that some of them are active in processes other than translation. In addition, there is evidence of tissue- and developmental-stage-specific expression for some of these proteins. These new findings point to an additional level of complexity in the translation initiation process. In this review, we analyze the latest advances concerning the functionality of members of the eIF4 families in eukaryotic organisms and discuss the implications of this in the context of our current understanding of regulation of the translation initiation process.

Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila

The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 were able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescued a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms were detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner.

Translation initiation factor eIF-4E from Drosophila: cDNA sequence and expression of the gene

A Drosophila melanogaster cDNA clone encoding the translation initiation factor eIF-4E was isolated and sequenced. The deduced polypeptide consists of 259 amino acids with a predicted molecular weight of 29,223. It shares 48%, 37% and 35% identity to its mammalian, yeast and wheat counterparts, respectively. Several residues (including eight tryptophans), which were shown to be critical for the function of mammalian and yeast eIF-4Es, are conserved in the Drosophila protein. Three transcripts of the eIF-4E gene were detected throughout Drosophila development.

Localization, structure and expression of the gene for translation initiation factor eIF-4E from Drosophila melanogaster

Drosophila melanogaster contains a single copy of the gene encoding translation eukaryotic initiation factor eIF-4E, which maps in the 67A2B1 region of chromosome 3L. A genomic clone containing the entire eIF-4E gene was isolated and sequenced. Comparison of this sequence with a cDNA previously obtained in our laboratory and 5′-RACE analysis revealed the existence of three mRNAs that are generated by alternative splicing of a primary transcript. All of them have different 5′untranslated leader regions. Two of the mRNAs encode the same eIF-4E polypeptide, whose sequence has been deduced from the cDNA clone. The third mRNA species contains a new open reading frame, possibly encoding another isoform of eIF-4E, which is very similar in size to the other but has a different N-terminal sequence. Several sequences which may be involved in the regulation of transcription initiation of the eIF-4E gene, except for a consensus TATA box, were found upstream of the putative transcription initiation sites. Expression of the eIF-4E gene is spatially and temporally controlled during embryonic development. It is ubiquitously expressed during embryogenesis but transcripts preferentially accumulate in certain tissues, particularly in the pole cells, at different developmental stages.

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5′ cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.

Different modes of translation for hid, grim and sickle mRNAs in Drosophila

Protein synthesis is inhibited during apoptosis. However, the translation of many mRNAs still proceeds driven by internal ribosome entry sites (IRESs). Here we show that the 5′UTR of hid and grim mRNAs promote translation of uncapped-mRNA reporters in cell-free embryonic extracts and that hid and grim mRNA 5′UTRs drive IRES-mediated translation. The translation of capped-reporters proceeds in the presence of cap competitor and in extracts where cap-dependent translation is impaired. We show that the endogenous hid and grim mRNAs are present in polysomes of heat-shocked embryos, indicating that cap recognition is not required for translation. In contrast, sickle mRNA is translated in a cap-dependent manner in all these assays. Our results show that IRES-dependent initiation may play a role in the translation of Drosophila proapoptotic genes and suggest a variety of regulatory pathways.

The distribution of eIF4E-family members across insecta

Insects are part of the earliest faunas that invaded terrestrial environments and are the first organisms that evolved controlled flight. Nowadays, insects are the most diverse animal group on the planet and comprise the majority of extant animal species described. Moreover, they have a huge impact in the biosphere as well as in all aspects of human life and economy; therefore understanding all aspects of insect biology is of great importance. In insects, as in all cells, translation is a fundamental process for gene expression. However, translation in insects has been mostly studied only in the model organism Drosophila melanogaster. We used all publicly available genomic sequences to investigate in insects the distribution of the genes encoding the cap-binding protein eIF4E, a protein that plays a crucial role in eukaryotic translation. We found that there is a diversity of multiple ortholog genes encoding eIF4E isoforms within the genus Drosophila. In striking contrast, insects outside this genus contain only a single eIF4E gene, related to D. melanogaster eIF4E-1. We also found that all insect species here analyzed contain only one Class II gene, termed 4E-HP. We discuss the possible evolutionary causes originating the multiplicity of eIF4E genes within the genus Drosophila.

Mextli Is a Novel Eukaryotic Translation Initiation Factor 4E-Binding Protein That Promotes Translation in Drosophila melanogaster

ABSTRACT Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5′-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

Cap binding-independent recruitment of eIF4E to cytoplasmic foci.

Eukaryotic translation initiation factor 4E (eIF4E) is required for cap-dependent initiation. In addition, eIF4E occurs in cytoplasmic foci such as processing bodies (PB) and stress granules (SG). We examined the role of key functional amino acid residues of eIF4E in the recruitment of this protein to cytoplasmic foci. We demonstrate that tryptophan residues required for mRNA cap recognition are not required for the recruitment of eIF4E to SG or PB. We show that a tryptophan residue required for protein-protein interactions is essential for the accumulation of eIF4E in granules. Moreover, we show, by the analysis of two Drosophila eIF4E isoforms, that the tryptophan residue is the common feature for eIF4E for the transfer of active mRNA from polysomes to other ribonucleoprotein particles in the cytoplasm. This residue resides in a putative interaction domain different than the eIF4E-BP domain. We conclude that protein-protein interactions rather than interactions with the mRNA are essential for the recruitment of eIF4E and for a putative nucleation function.

Erratum: Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila (Mechanisms of Development (2005) 122 (529-543) DOI: 10.1016/j.mod.2004.11.011)

a Max-Planck-Institut für Biophysikalische Chemie, Abt. Molekulare Biologie, Am Fassberg 11, 37077 Göttingen, Germany b Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, 3012 Bern, Switzerland c Centro de Biologı́a Molecular “Severo Ochoa”, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain d Max-Planck-Institut für Biophysikalische Chemie, Abt. Zelluläre Biochemie, Am Fassberg 11, 37077 Göttingen, Germany e School of Life Sciences, University of Dundee, Wellcome Trust Biocenter, Dundee DD1 5EH, Scotland, UK f Zentrum Anatomie, Abt. Anatomie-Embryologie, Georg-August-Universität Göttingen, Kreuzbergring 36, 37075 Göttingen, Germany