EIF4G-driven translation initiation of downstream ORFs in mammalian cells.
ABSTRACT: Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.
Project description:Resistance of translation of some eukaryotic messenger RNAs (mRNAs) to inactivation of the cap-binding factor eIF4E under unfavorable conditions is well documented. To date, it is the mechanism of internal ribosome entry that is predominantly thought to underlay this stress tolerance. However, many cellular mRNAs that had been considered to contain internal ribosome entry sites (IRESs) failed to pass stringent control tests for internal initiation, thus raising the question of how they are translated under stress conditions. Here, we show that inserting an eIF4G-binding element from a virus IRES into 5'-UTRs of strongly cap-dependent mRNAs dramatically reduces their requirement for the 5'-terminal m(7)G-cap, though such cap-independent translation remains dependent on a vacant 5'-terminus of these mRNAs. Importantly, direct binding of eIF4G to the 5'-UTR of mRNA makes its translation resistant to eIF4F inactivation both in vitro and in vivo. These data may substantiate a new paradigm of translational control under stress to complement IRES-driven mechanism of translation.
Project description:Picornaviruses use internal ribosome entry sites (IRESs) to translate their genomes into protein. A typical feature of these IRESs is their ability to bind directly to the eukaryotic initiation factor (eIF) 4G component of the eIF4F cap-binding complex. Remarkably, the hepatitis A virus (HAV) IRES requires eIF4E for its translation, but no mechanism has been proposed to explain this. Here we demonstrate that eIF4E regulates HAV IRES-mediated translation by two distinct mechanisms. First, eIF4E binding to eIF4G generates a high-affinity binding conformation of the eIF4F complex for the IRES. Second, eIF4E binding to eIF4G strongly stimulates the rate of duplex unwinding by eIF4A on the IRES. Our data also reveal that eIF4E promotes eIF4F binding and increases the rate of restructuring of the poliovirus (PV) IRES. This provides a mechanism to explain why PV IRES-mediated translation is stimulated by eIF4E availability in nuclease-treated cell-free extracts. Using a PV replicon and purified virion RNA, we also show that eIF4E promotes the rate of eIF4G cleavage by the 2A protease. Finally, we show that cleavage of eIF4G by the poliovirus 2A protease generates a high-affinity IRES binding truncation of eIF4G that stimulates eIF4A duplex unwinding independently of eIF4E. Therefore, our data reveal how picornavirus IRESs use eIF4E-dependent and -independent mechanisms to promote their translation.
Project description:We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5' untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5' UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5' UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.
Project description:Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5'UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2? and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.
Project description:Translation is downregulated in response to a variety of moderate stresses, including serum deprivation, hyperosmolarity and ionizing radiation. The cytostatic p21-activated protein kinase 2 (Pak2)/gamma-PAK is activated under the same stress conditions. Expression of wild-type Pak2 in cells and addition of Pak2 to reticulocyte lysate inhibit translation, while kinase-inactive mutants have no effect. Pak2 binds to and phosphorylates initiation factor (eIF)4G, which inhibits association of eIF4E with m(7)GTP, reducing initiation. The Pak2-binding site maps to the region on eIF4G that contains the eIF4E-binding site; Pak2 and eIF4E compete for binding to this site. Using an eIF4G-depleted reticulocyte lysate, reconstitution with mock-phosphorylated eIF4G fully restores translation, while phosphorylated eIF4G reduces translation to 37%. RNA interference releases Pak2-induced inhibition of translation in contact-inhibited cells by 2.7-fold. eIF4G mutants of the Pak2 site show that S896D inhibits translation, while S896A has no effect. Activation of Pak2 in response to hyperosmotic stress inhibits cap-dependent, but not IRES-driven, initiation. Thus, a novel pathway for mammalian cell stress signaling is identified, wherein activation of Pak2 leads to inhibition of cap-dependent translation through phosphorylation of eIF4G.
Project description:The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between ?-sheet2 (L60-T68) and ?-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending ?-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.
Project description:Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.
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:Elevated eukaryotic initiation factor 4E (eIF4E) levels frequently occur in a variety of human cancers. Overexpression of eIF4E promotes cellular transformation by selectively increasing the translation of proliferative and prosurvival mRNAs. These mRNAs possess highly structured 5'-UTRs that impede ribosome recruitment and scanning, yet the mechanism for how eIF4E abundance elevates their translation is not easily explained by its cap-binding activity. Here, we show that eIF4E possesses an unexpected second function in translation initiation by strongly stimulating eukaryotic initiation factor 4A (eIF4A) helicase activity. Importantly, we demonstrate that this activity promotes mRNA restructuring in a manner that is independent of its cap-binding function. To explain these findings, we show that the eIF4E-binding site in eukaryotic initiation factor 4G (eIF4G) functions as an autoinhibitory domain to modulate its ability to stimulate eIF4A helicase activity. Binding of eIF4E counteracts this autoinhibition, enabling eIF4G to stimulate eIF4A helicase activity. Finally, we have successfully separated the two functions of eIF4E to show that its helicase promoting activity increases the rate of translation by a mechanism that is distinct from its cap-binding function. Based on our results, we propose that maintaining a connection between eIF4E and eIF4G throughout scanning provides a plausible mechanism to explain how eIF4E abundance selectively stimulates the translation of highly structured proliferation and tumor-promoting mRNAs.
Project description:Control of translation in eukaryotes is complex, depending on the binding of various factors to mRNAs. Available data for subsets of mRNAs that are translationally up- and down-regulated in yeast eIF4E-binding protein (4E-BP) deletion mutants are coupled with reported mRNA secondary structure measurements to investigate whether 5'-UTR secondary structure varies between the subsets. Genes with up-regulated translational efficiencies in the caf20? mutant have relatively high averaged 5'-UTR secondary structure. There is no apparent wide-scale correlation of RNA-binding protein preferences with the increased 5'-UTR secondary structure, leading us to speculate that the secondary structure itself may play a role in differential partitioning of mRNAs between eIF4E/4E-BP repression and eIF4E/eIF4G translation initiation. Both Caf20p and Eap1p contain stretches of positive charge in regions of predicted disorder. Such regions are also present in eIF4G and have been reported to associate with mRNA binding. The pattern of these segments, around the canonical eIF4E-binding motif, varies between each 4E-BP and eIF4G. Analysis of gene ontology shows that yeast proteins containing predicted disordered segments, with positive charge runs, are enriched for nucleic acid binding. We propose that the 4E-BPs act, in part, as differential, flexible, polyelectrostatic scaffolds for mRNAs.