Paola Londei

The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis

The mammalian target of rapamycin, mTOR, regulates cell growth and proliferation. Here we show that the initiation factor of translation (eIF-4E), a downstream effector of mTOR, has oncogenic effects in vivo and cooperates with c-Myc in B-cell lymphomagenesis. We found that c-Myc overrides eIF-4E-induced cellular senescence, whereas eIF-4E antagonizes c-Myc-dependent apoptosis in vivo. Our results implicate activation of eIF-4E as a key event in oncogenic transformation by phosphoinositide-3 kinase and Akt.

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet‐tRNAfMet. Leaderless λ cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5′‐terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of λ cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S–fMet‐tRNAfMet–IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless λ cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

Ribosome recycling depends on a mechanistic link between the FeS cluster domain and a conformational switch of the twin-ATPase ABCE1

Despite some appealing similarities of protein synthesis across all phyla of life, the final phase of mRNA translation has yet to be captured. Here, we reveal the ancestral role and mechanistic principles of the newly identified twin-ATPase ABCE1 in ribosome recycling. We demonstrate that the unique iron-sulfur cluster domain and an ATP-dependent conformational switch of ABCE1 are essential both for ribosome binding and recycling. By direct (1∶1) interaction, the peptide release factor aRF1 is shown to synergistically promote ABCE1 function in posttermination ribosome recycling. Upon ATP binding, ABCE1 undergoes a conformational switch from an open to a closed ATP-occluded state, which drives ribosome dissociation as well as the disengagement of aRF1. ATP hydrolysis is not required for a single round of ribosome splitting but for ABCE1 release from the 30S subunit to reenter a new cycle. These results provide a mechanistic understanding of final phases in mRNA translation.

Cis-acting signals controlling translational initiation in the thermophilic archaeon Sulfolobus solfataricus

In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis‐acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine–Dalgarno (SD)‐like ribosome‐binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5′ untranslated region altogether, thereby creating a ‘leaderless’ mRNA. This suggests that ‘leaderless’ initiation operates by a default mechanism that does not require a specific mRNA–rRNA interaction and may be common to all three primary domains of life.

Insensitivity of archaebacterial ribosomes to protein synthesis inhibitors. Evolutionary implications.

The effect on Sulfolobus solfataricus (an extremely thermoacidophilic archaebacterium) of selected inhibitors affecting reactions of the polypeptide elongation cycle has been tested by using poly(U) and poly(UG) directed cell‐free systems. The results reveal a unique pattern of antibiotic sensitivity of Sulfolobus ribosomes with an inhibitory effect observed for only three of 60 compounds tested. Through comparison with suitable eubacterial and eukaryotic cell‐free systems the insensitivity of Sulfolobus ribosomes to most inhibitors of protein synthesis appears to reflect a phylogenetic distinction of ribosome structure, rather than the high temperature conditions of the Sulfolobus assay system. In this respect ribosomes of thermoacidophilic archaebacteria differ not only from their eubacterial and eukaryotic counterparts, but also from ribosomes of archaebacteria belonging to the methanogenic‐halophilic branch of the ‘third’ kingdom. The evolutionary implications of these findings are discussed.

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation

In this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with Shine–Dalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (65–70°C) and without the aid of initiator tRNA (tRNAi) or any factor. ‘Toeprinting’ assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5′‐untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codon–anticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD‐dependent initiation, resembling the pathway prevalent in present‐day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas ‘leaderless’ initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.

Translation initiation factor a/eIF2(-γ) counteracts 5′ to 3′ mRNA decay in the archaeon Sulfolobus solfataricus

The trimeric translation initiation factor a/eIF2 of the crenarchaeon Sulfolobus solfataricus is pivotal for binding of initiator tRNA to the ribosome. Here, we present in vitro and in vivo evidence that the a/eIF2 γ-subunit exhibits an additional function with resemblance to the eukaryotic cap-complex. It binds to the 5′-triphosphate end of mRNA and protects the 5′ part from degradation. This unprecedented capacity of the archaeal initiation factor further indicates that 5′ → 3′ directional mRNA decay is a pathway common to all domains of life.

The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double‐ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so‐called CCT‐TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exists that the archaeal chaperonins are implicated in protein folding, much remains to be learned about their precise cellular function. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus is an RNA‐binding protein that interacts specifically in vivo with the 16S rRNA and participates in the maturation of its 5′ extremity in vitro. We further show that the chaperonin binds RNA as the native heterooligomeric complex and that RNA binding and processing are inhibited by ATP. These results agree with previous reports indicating a role for the bacterial/organellar chaperonins in RNA protection or processing and suggest that all known chaperonin families share specific and evolutionarily ancient functions in RNA metabolism.

Translation initiation in Archaea: conserved and domain-specific features

Initiation is a critical step in translation, during which the ribosome lands on the start codon and sets the correct reading frame for mRNA decoding. The rate and efficiency of translation are largely determined by initiation, which is therefore the preferred target of translation regulation mechanisms. Initiation has incurred an extensive evolutionary divergence among the primary domains of cell descent. The Archaea, albeit prokaryotes, have an initiation mechanism and apparatus more complex than those of the Bacteria; the molecular details of archaeal initiation are just beginning to be unravelled. The most notable aspects of archaeal initiation are the presence of two, perhaps three, distinct mechanisms for mRNA-ribosome interaction and the presence of a relatively large set of IFs (initiation factors), several of which are shared exclusively with the Eukarya. Among these, the protein termed a/eIF2 (archaeal/eukaryotic IF2) and aIF6 (archaeal IF6) are of special interest, since they appear to play key regulatory roles in the Eukarya. Studies of the function of these factors in Archaea have uncovered new features that will help to elucidate their conserved and domain-specific functions.