Biomedical subjects

Greco Hernández

Publications and source records attributed to Greco Hernández.

6 recordsLinked to original sources

PubMed2005

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.

Animals↗

PubMed2004

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.

Aging↗

PubMed2004

Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster.

We have identified the initiation factor 4A (eIF4A) in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A.

Alternative Splicing↗

PubMed2004

Two functionally redundant isoforms of Drosophila melanogaster eukaryotic initiation factor 4B are involved in cap-dependent translation, cell survival, and proliferation.

Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome. DrosophilaeIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs. Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle. The proteins are cytoplasmic with polarized distribution. The two isoforms bind RNA with the same affinity. eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system. RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed. Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.

Amino Acid Sequence↗

PubMed2004

Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos.

Translation is a sensitive regulatory step during cellular stress and the apoptosis response. Under such conditions, cap-dependent translation is reduced and internal ribosome entry site (IRES)-dependent translation plays a major role. However, many aspects of how mRNAs are translated under stress remain to be elucidated. Here we report that reaper mRNA, a pro-apoptotic gene from Drosophila melanogaster, is translated in a cap-independent manner. In Drosophila mutant embryos devoid of the eukaryotic initiation factor 4E (eIF4E), reaper transcription is induced and apoptosis proceeds. In vitro translation experiments using wild-type and eIF4E mutant embryonic extracts show that reporter mRNA bearing reaper 5' untranslated region (UTR) is effectively translated in a cap-independent manner. The 5'UTR of reaper exhibits a high degree of similarity with that of Drosophila heat shock protein 70 mRNA, and both display IRES activity. Studies of mRNA association to polysomes in embryos indicate that both reaper and heat shock protein 70 mRNAs are recruited to polysomes under apoptosis or thermal stress. Our data suggest that heat shock protein 70 and reaper, two antagonizing factors in apoptosis, use a similar mechanism for protein synthesis.

5' Untranslated Regions↗

PubMed2003

Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase.

Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity.

Amino Acid Sequence↗