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.

Eukaryotic Initiation Factor 4G Suppresses Nonsense-Mediated mRNA Decay by Two Genetically Separable Mechanisms

Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.

Unique modifications of translation elongation factors

Covalent modifications of proteins often modulate their biological functions or change their subcellular location. Among the many known protein modifications, three are exceptional in that they only occur on single proteins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation. For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol. We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms.

Unique posttranslational modifications in eukaryotic translation factors and their roles in protozoan parasite viability and pathogenesis.

Protozoan parasites are one of the major causes of diseases worldwide. The vector transmitted parasites exhibit complex life cycles involving interactions between humans, protozoa, and arthropods. In order to adapt themselves to the changing microenvironments, they have to undergo complex morphological and metabolic changes. These changes can be brought about by expressing a new pool of proteins in the cell or by modifying the existing repertoire of proteins via posttranslational modifications (PTMs). PTMs involve covalent modification and processing of proteins thereby modulating their functions. Some of these changes may involve PTMs of parasite proteins to help the parasite survive within the host and the vector. Out of many PTMs known, three are unique since they occur only on single proteins: ethanolamine phosphoglycerol (EPG) glutamate, hypusine and diphthamide. These modifications occur on eukaryotic elongation factor 1A (eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elongation factor 2 (eEF2), respectively. Interestingly, the proteins carrying these unique modifications are all involved in the elongation steps of translation. Here we review these unique PTMs, which are well conserved in protozoan parasites, and discuss their roles in viability and pathogenesis of parasites. Characterization of these modifications and studying their roles in physiology as well as pathogenesis will provide new insights in parasite biology, which may also help in developing new therapeutic interventions.

The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation

PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

The quassinoid derivative NBT-272 targets both the AKT and ERK signaling pathways in embryonal tumors

The quassinoid analogue NBT-272 has been reported to inhibit MYC, thus warranting a further effort 7to better understand its preclinical properties in models of embryonal tumors (ET), a family of childhood malignancies sharing relevant biological and genetic features such as deregulated expression of MYC oncogenes. In our study, NBT-272 displayed a strong antiproliferative activity in vitro that resulted from the combination of diverse biological effects, ranging from G(1)/S arrest of the cell cycle to apoptosis and autophagy. The compound prevented the full activation of both eukaryotic translation initiation factor 4E (eIF4E) and its binding protein 4EBP-1, regulating cap-dependent protein translation. Interestingly, all responses induced by NBT-272 in ET could be attributed to interference with 2 main proproliferative signaling pathways, that is, the AKT and the MEK/extracellular signal-regulated kinase pathways. These findings also suggested that the depleting effect of NBT-272 on MYC protein expression occurred via indirect mechanisms, rather than selective inhibition. Finally, the ability of NBT-272 to arrest tumor growth in a xenograft model of neuroblastoma plays a role in the strong antitumor activity of this compound, both in vitro and in vivo, with its potential to target cell-survival pathways that are relevant for the development and progression of ET.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

Characterization of Phosphorylation- and RNA-Dependent UPF1 Interactors by Quantitative Proteomics

Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1’s phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Mechanistic aspects of mRNA targeting for nonsense-mediated mRNA decay in human cells

The nonsense-mediated mRNA decay (NMD) pathway is best known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with ORF-truncating premature termination codons (PTCs), but a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD substrates. We try to decipher the mechanism of mRNA targeting to the NMD pathway in human cells. Recruitment of the conserved RNA-binding helicase UPF1 to target mRNAs has been reported to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. We have transcriptome-wide determined the UPF1 binding sites by individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation. We detected a strongly enriched association of UPF1 with 3’ UTRs in undisturbed, translationally active cells. After translation inhibition, a significant increase in UPF1 binding to coding sequence (CDS) was observed, indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This suggests that the decision to trigger NMD occurs after association of UPF1 with mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. In a second recent study, we re-visited the reported restriction of NMD in mammals to the ‘pioneer round of translation’, i.e. to cap-binding complex (CBC)-bound mRNAs. The limitation of mammalian NMD to early rounds of translation would indicate a – from an evolutionary perspective – unexpected mechanistic difference to NMD in yeast and plants, where PTC-containing mRNAs seem to be available to NMD at each round of translation. In contrast to previous reports, our comparison of decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells revealed that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.