Biomedical subjects

Michael Altmann

Publications and source records attributed to Michael Altmann.

7 recordsLinked to original sources

PubMed2006

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

3' Untranslated Regions↗

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

Crystal structure of yeast Ypr118w, a methylthioribose-1-phosphate isomerase related to regulatory eIF2B subunits.

Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the alpha-, beta-, and delta-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.

Aldose-Ketose Isomerases↗

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

Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene.

The 5'-region of the TIF4631 gene of Saccharomyces cerevisiae (encoding the translation initiation factor eIF4G1) was reported earlier to harbor a very active internal ribosome entry site (IRES) allowing for internal initiation of translation of TIF4631 mRNA. Here, we report the presence of a promoter in the region -112 to -36 relative to the translation initiation codon of the TIF4631 gene. This promoter stimulates transcription from a start site at position -36 and generates an mRNA that is actively translated in vitro and able to sustain growth of yeast cells in vivo as the only source of eIF4G. The data show that the IRES activity reported earlier is due to this promoter. On the contrary, the presumed IRES represents a strongly inhibitory element for translation in vitro.

5' Untranslated Regions↗

PubMed2003

Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells.

The [URE3] phenotype in Saccharomyces cerevisiae is caused by the inactive, altered (prion) form of the Ure2 protein (Ure2p), a regulator of nitrogen catabolism. Ure2p has two functional domains: an N-terminal domain necessary and sufficient for prion propagation and a C-terminal domain responsible for nitrogen regulation. We show here that the mRNA encoding Ure2p possesses an IRES (internal ribosome entry site). Internal initiation leads to the synthesis of an N-terminally truncated active form of the protein (amino acids 94-354) lacking the prion-forming domain. Expression of the truncated Ure2p form (94-354) mediated by the IRES element cures yeast cells of the [URE3] phenotype. We assume that the balance between the full-length and truncated (94-354) Ure2p forms plays an important role in yeast cell physiology and differentiation.

Animals↗

PubMed2003

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae.

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.

Base Sequence↗