Cellular mRNA recruits the ribosome via eIF3-PABP bridge to initiate internal translation.

IRES-mediated translation of key cell fate regulating genes has been implicated in tumorigenesis. Concerted action of canonical eukaryotic initiation factors and IRES transacting factors (ITAFs) was shown to regulate cellular IRES mediated translation; however, the precise molecular mechanism of ribosome recruitment to cellular IRESes remains unclear. Here we show that the X-linked inhibitor of apoptosis (XIAP) IRES operates in an evolutionary conserved viral like mode and the structural integrity, particularly in the vicinity of AUG, is critical for ribosome recruitment. The binding of eIF3 together with PABP potentiates ribosome recruitment to the IRES. Our data support the model in which eIF3 binds directly to the XIAP IRES RNA in a structure-dependent manner and acts as a scaffold for IRES RNA, PABP and the 40S ribosome.

Assembly of eIF3 Mediated by Mutually Dependent Subunit Insertion.

Eukaryotic initiation factor 3 (eIF3), an essential multi-protein complex involved in translation initiation, is composed of 12 tightly associated subunits in humans. While the overall structure of eIF3 is known, the mechanism of its assembly and structural consequences of dysregulation of eIF3 subunit expression seen in many cancers is largely unknown. Here we show that subunits in eIF3 assemble into eIF3 in an interdependent manner. Assembly of eIF3 is governed primarily by formation of a helical bundle, composed of helices extending C-terminally from PCI-MPN domains in eight subunits. We propose that, while the minimal subcomplex of human-like eIF3 functional for translation initiation in cells consists of subunits a, b, c, f, g, i, and m, numerous other eIF3 subcomplexes exist under circumstances of subunit over- or underexpression. Thus, eIF3 subcomplexes formed or "released" due to dysregulated subunit expression may be determining factors contributing to eIF3-related cancers.

Human-like eukaryotic translation initiation factor 3 from Neurospora crassa.

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13(th) subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.

Architecture of human translation initiation factor 3.

Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans.

Use of ribozyme cleavage kinetics to measure salt-induced changes in solution pH.

Several ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid-base catalysis. Interpreting the apparent pK(a) values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS ribozyme as a "biological pH meter" to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a.

Several small ribozymes employ general acid-base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK (a) values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK (a) values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid-base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid-base catalysis indicate that k (1), the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK (a). The inference that k (1) is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK (a) is that of the general acid and the higher pK (a) is that of the general base.

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method.

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme.

A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec(-1), similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pK(a)s of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D(2)O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH- and D(2)O-independent cleavage approximately 10(3)-fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate approximately 10(3)-fold, but the A756C mutant retains pH- and D(2)O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.