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Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites.

ABSTRACT: Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3' borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2-4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3' borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J-K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.

SUBMITTER: de Breyne S

PROVIDER: S-EPMC2695064 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites.

de Breyne Sylvain S Yu Yingpu Y Unbehaun Anett A Pestova Tatyana V TV Hellen Christopher U T CU

Proceedings of the National Academy of Sciences of the United States of America 20090522 23

Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3' borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2-4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical comp ...[more]

PMID: 19470487

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Project description:As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5' untranslated region (5'-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5'-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5'-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPbeta. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5'-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5'-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.

Project description:In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5' untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5'-proximal codons of CHV1 as well as the 5' UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5' untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

Dataset Information

Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites.

Publications

Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites.

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