Eukaryotic translation initiation machinery can operate in a prokaryotic-like mode without eIF2

Unlike prokaryotes, a specialized eukaryotic initiation factor 2 (eIF2), in the form of the ternary complex eIF2*GTP*Met-tRNAiMet is utilized to deliver the initiator tRNA to the ribosome within all eukaryotic cells1. Phosphorylation of eIF2 is known to be central to the global regulation of protein synthesis under stress conditions and infection2. Another distinctive feature of eukaryotic translation is scanning of mRNA 5'-leaders, whose origin in evolution may be relevant to the appearance of eIF2 in eukaryotes. Translation initiation on the hepatitis C virus (HCV) internal ribosome entry site (IRES) occurs without scanning3,4. Whether these unique features of the HCV IRES account for the formation of the final 80S initiation complex is unknown. Here we show that the HCV IRES-directed translation can occur without either eIF2 or its GTPase activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a prokaryotic-like pathway which involves eIF5B, the analogue of bacterial IF25,6, instead of eIF2. This switch from a eukaryotic-like mode of AUG selection to a "bacterial" one occurs when eIF2 is inactivated by phosphorylation, a way with which host cells counteract infection. The relative resistance of HCV IRES-directed translation to eIF2 phosphorylation may represent one more line of defense used by this virus against host antiviral responses and can contribute to the well known resistance of HCV to interferon based therapy

The 5′ untranslated region of Apaf-1 mRNA directs translation under apoptosis conditions via a 5′ end-dependent scanning mechanism

AbstractWe have previously shown that translation driven by the 5′ UTR of Apaf-1 mRNA is relatively efficient in the absence of m7G-cap, but no IRES is involved. Nevertheless, it may be speculated that a “silent” IRES is activated under apoptosis conditions. Here, we show that translation of the mRNA with the Apaf-1 5′ UTR is relatively resistant to apoptosis induced by etoposide when eIF4E is sequestered by 4E-BP and eIF4G is partially cleaved. However, translation under these conditions remains governed by 5′ end-dependent scanning. We hypothesize that the observed phenomenon is based on the intrinsic low cap-dependence of the Apaf-1 5′ UTR

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed

Differential contribution of the m7G-cap to the 5′ end-dependent translation initiation of mammalian mRNAs

Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated

Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2. Nature Structural & Molecular Biology.

Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA i Met , is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2-and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions. The bacterial translation initiation mechanism uses only three initiation factors: IF1, IF2 and IF3 (ref. 1). In recent years, their functional analogs have been identified in eukaryotic cells; namely, eIF1A, eIF5B and eIF1, respectively 2 . In spite of this similarity, the molecular mechanisms of translation initiation in bacteria and eukaryotes are believed to be significantly different. The principal distinctive feature of eukaryotic translation is the scanning of the mRNA 5¢ untranslated regions (5¢ UTRs) 3 . The initial recognition of the mRNA occurs through the interaction between the 5¢ terminal m 7 G cap and the cap binding complex eIF4F. The ribosome is loaded onto the 5¢ end of the mRNA and is then thought to migrate downstream (to 'scan') to locate an AUG codon in a favorable context. The factors eIF1 and eIF1A are indispensable for scanning. The helicase activity of eIF4F, stimulated by eIF4B, unwinds secondary structures within the mRNA during scanning. Another distinctive feature is the presence of eIF2, which, in the form of the ternary complex eIF2-GTP-Met-tRNA i Met , brings the initiator tRNA to the ribosome 3 . There is no analog of eIF2 in bacteria. The role of eIF2 is broader than just delivering MettRNA i Met to the ribosome, as phosphorylation of eIF2 is known to be central to the global regulation of protein synthesis under stress conditions and during virus infection 4 . This complicated machinery operates not only in the case of standard cap-dependent mRNAs but also with mRNAs whose translation initiation uses the binding of ribosomes to IRESs. These structural elements bind diverse components of the translation initiation apparatus and thereby direct ribosome binding to the vicinity of the initiation codon. Although most of the well-studied IRESs from picornaviruses do not require the cap binding factor eIF4E, their molecular mechanisms of initiation are no simpler than that for standard cap-dependent mRNAs, and they frequently require additional mRNA binding proteins 5 . The only exception, among IRES elements using methionine-based translation initiation, are the IRES elements of HCV RNA and the HCV-like IRESs from some other flavi-and picornaviruses. They bind directly to the 40S ribosomal subunit and position it close to the AUG codon so that no scanning is required, and this strongly resembles the prokaryotic mode of AUG selection. Consequently, the cap binding complex eIF4F and the scanning factors eIF1 and eIF1A are not required. For this type of IRES element, the 48S translation initiation complex can be formed with just eIF2-GTP-Met-tRNA i Met (refs. 6,7). It was intriguing to know whether the unique features of the HCV IRES are also involved in the formation of the final 80S initiation complex. Here we have explored the mechanism of 80S complex formation on the HCV IRES using totally purified components of the translational apparatus from mammalian cells. We have found that, in addition to the conventional eIF2-dependent mode of translation initiation, the HCV IRES can use a bacterial-like, eIF2-independent mechanism. In this case, it is apparently eIF5B, the homolog of prokaryotic IF2 (ref. 8), that promotes initiator tRNA binding to the ribosomal P site. Inactivation of eIF2 by phosphorylation, in response to various treatments, has a much smaller effect on HCV IRES-mediated translation than on cap-dependent translation. The switch from the eukaryotic to a bacterial-like mode of translation initiation occurs when eIF2 is inactivated. RESULTS 80S complex can be formed on the HCV IRES without eIF2 Previous work showed that the 48S initiation complex can be formed on the HCV IRES with 40S subunits and the eIF2-GTP-Met-tRNA i Met complex only. The factors eIF1 and eIF4 were dispensable 6 . The role of eIF3 remained unclear; it was suggested to be involved in the next step of initiation. As eIF2 was the crucial component for 48S comple

The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

RelE/RelB is a well-characterized toxin–anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making “RelE printing” a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site

In vitro activity of human translation initiation factor eIF4B is not affected by phosphomimetic amino acid substitutions S422D and S422E.

Eukaryotic translation initiation factor eIF4B is necessary for ribosomal scanning through structured mRNA leaders. In higher eukaryotes, eIF4B serves as a downstream effector of several signaling pathways. In response to mitogenic stimuli, eIF4B undergoes multiple phosphorylations which are thought to regulate its activity. Recently, Ser422 was identified as a predominant site for human eIF4B phosphorylation via several signaling pathways, and phosphomimetic amino acid substitutions S422D or S422E were shown to activate eIF4B in living cells. However, stimulatory role of these modifications has never been analyzed directly. Here, using both mammalian reconstituted translation initiation assay and complete cell-free translation system, we perform a comparison of recombinant eIF4B derivatives with the wild type recombinant protein, and do not find any difference in their activities. On the contrary, native eIF4B purified from HeLa cells reveals significantly higher activity in both assays. Thus, the effects of S422D and S422E substitutions on eIF4B activity in living cells observed previously either require some other protein modification(s), or may only be manifested in an intact cell. Our study raises the question on whether the phosphorylation of Ser422 is sufficient for eIF4B activation observed upon mitogenic stimulation