Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.
Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182–mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.
Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5 ′ -terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5 ′ end of mRNA, implying that Met-tRNA i Met inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNA i Met can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m 7 G cap specifically interacts with eIF3l.[Keywords: eukaryotic translation initiation; eIF4E; eIF4F; m 7 G; eIF3l; 40S ribosomal subunit]Supplemental material is available for this article.Received April 7, 2016; revised version accepted June 1, 2016.Translation initiation on the majority of mammalian cellular mRNAs occurs by the scanning mechanism (Jackson et al. 2010). First, eukaryotic initiation factor 2 (eIF2), GTP, and Met-tRNA i Met form a ternary complex (eIF2-TC), which, with the multisubunit eIF3 and monomeric eIF1 and eIF1A, binds to the 40S ribosomal subunit, yielding a 43S preinitiation complex. Attachment of the 43S complex to capped mRNA is mediated by eIF4A, eIF4B, and eIF4F. eIF4F comprises the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A (which also exists in a free form), and the eIF4G scaffold, which interacts with the other two subunits as well as eIF3. eIF4A's helicase activity is stimulated by both eIF4G and eIF4B. Group 4 eIFs cooperatively unwind the cap-proximal region of mRNA, preparing it for attachment of 43S complexes, which is promoted by the eIF4G-eIF3 interaction. After attachment, 43S complexes scan to the first AUG codon in a favorable nucleotide context, where they form 48S initiation complexes with established codonanti-codon base-pairing. Group 4 eIFs also assist 43S complexes during scanning. Scanning on mRNAs with highly structured 5 ′ untranslated regions (UTRs) additionally requires DHX29, a DExH-box protein that interacts directly with 40S subunits. eIF1, in cooperation with eIF1A, ensures the fidelity of initiation codon selection, discriminating against initiation at non-AUG codons and AUGs that are too close to the 5 ′ end of mRN...
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