The stage of development between birth and weaning in mammals is a period of very rapid growth that is crucial for the long-term well-being of the animal. The rate of protein deposition in neonatal animals is very high because dietary protein is efficiently utilized to increase body protein mass. Our studies in neonatal pigs have shown that this high efficiency of protein deposition is largely due to the marked increase in protein synthesis after feeding, and this response is particularly profound in the skeletal muscle. The enhanced stimulation of muscle protein synthesis in neonates after feeding is independently mediated by the rise in insulin and amino acids and this response declines with age. Intracellular signaling components that respond to the postprandial rise in amino acids and insulin have been identified and their activation has been shown to be elevated in skeletal muscle of neonatal pigs after a meal and to decrease with development. The enhanced activation of these components in the amino acid and insulin signaling pathways in neonatal muscle contributes to the high rate of muscle protein synthesis and rapid gain in skeletal muscle mass in newborn pigs, which are essential determinants of efficient growth during development.

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis—the highly conserved 3′ end of 16S rRNA (aSD) complementary to the Shine–Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving β-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the β-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5′ untranslated region, it can ensure a substantial enhancing effect on translation.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

Eukaryotic initiation factor (eIF) 4A functions as a subunit of the initiation factor complex eIF4F, which mediates the binding of mRNA to the ribosome. eIF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of eIF4A during translation initiation is to unwind the mRNA secondary structure in the 5′ UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that eIF4A is also required for the translation of mRNAs possessing minimal 5′ UTR secondary structure. Were this hypothesis correct, the requirement for eIF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian eIF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5′ untranslated region are more susceptible to inhibition by the eIF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of eIF4F subunits and eIF4B to the mRNA cap structure is dramatically reduced by the eIF4A mutant and RNA secondary structure. Finally, the eIF4A mutant forms a more stable complex with eIF4G, as compared to the wild-type eIF4A, thus explaining the mechanism by which substoichiometric amounts of mutant eIF4A inhibit translation.

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5′ untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA[bull ]40S[bull ]eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

The cap-binding complex eIF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: eIF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865–4874). Here we exploit this approach to assess which of the other subunits of eIF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the λ phage antiterminator protein N to generate the chimeric proteins λ4A, λ4E-102 (abolished cap binding), and λ4E-73–102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific λ-N binding site (boxB) in the intercistronic space. We show that λ4E-102, but neither the double mutant λ4E-73–102 nor λ4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all λ-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, λ4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein–RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1–2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3′ region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

In eubacteria, base pairing between the 3′ end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5′ untranslated region (5′ UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5′ of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5′ UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3′ domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA–rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5′-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3′-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5′- and 3′-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5′ nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5′ NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5′ terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5′ NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5′ sequences and/or acquisition of novel 5′ sequences such that the 5′ terminal 3–4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5′ NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals.

The cauliflower mosaic virus (CaMV) 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600-nt leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and packaging. We have recently documented that the 35 S RNA leader adopts an elongated hairpin conformation and additional higher-order structures, a long-range pseudoknot and a dimer. Alternative structures might coexist, probably fulfilling specialized functions. In this paper, we analyze the biological significance of the elongated hairpin structure. We have introduced a spectrum of large deletions and small substitutions in the 35 S RNA leader and characterized their impact on the structure by temperature gradient gel electrophoresis. This analysis showed that the elongated hairpin consists of three sections of different stability (stem section I, II, and III). The overall secondary structure is relatively stable in the range of 10–32°C. It melts between 32 and 38°C in a manner indicating that the most stable base pairing occurs at the base of the elongated hairpin (stem section I). Mutations that destabilize stem section I decrease both the melting temperature of the leader and the expression in vitro and in vivo of the downstream CAT-reporter gene. Compensatory mutations restoring the stable elongated hairpin upregulate the translation efficiency. Our results demonstrate that the regulation of translation of the CaMV 35 S RNA is mediated by a stable hairpin in the leader.