Recruitment of human eIF4G1 or DAP5 to the 5’ untranslated regions of a subset of cellular mRNAs drives cap-independent translation

During unfavorable cellular conditions (e.g., tumor hypoxia, viral infection, nutrient deprivation, etc.), the canonical, cap-dependent translation initiation pathway in human cells is suppressed by sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, by 4E-binding proteins. Additionally, the expression levels of eIF4G and its cellular homolog, death associated protein 5 (DAP5), are elevated. Under these conditions, a subset of cellular mRNAs, including many encoding proteins with important roles in human health and disease, (e.g. HIF-1α, FGF-9, and p53) is translated in a cap-independent manner. Despite their physiological importance, however, the molecular mechanisms underlying cap-independent initiation of this subset of cellular mRNAs remain unknown. Here, we have used fluorescence anisotropy-based equilibrium binding assays developed in our laboratories to demonstrate that an N-terminal truncated form of human eIF4G1 that cannot interact with eIF4E (ΔN-4G1) or DAP5 directly bind to the 5’ untranslated regions (UTRs) of these mRNAs. Specifically, we have measured the differential affinities with which ΔN-4G1 and DAP5 interact with the 5’ UTRs of four distinct members of this subset of cellular mRNAs. Using a luciferase-based gene expression reporter assay, we further demonstrate that these same 5’ UTRs can promote cap-independent initiation in an ΔN-4G1 or DAP5-dependent manner in a rabbit reticulocyte lysate-based in vitro translation system. Integrating the results of our quantitative binding- and in vitro translation studies, we propose a model specifying how a subset of cellular mRNAs switch from cap-dependent to cap-independent modes of translation initiation Significance Under cellular stress conditions (e.g., tumor hypoxia, viral infection, etc.), the canonical, cap-dependent initiation of eukaryotic protein synthesis, or translation, of a subset of cellular mRNAs switches to a cap-independent mechanism. Despite the important role they play in human health and disease, the mechanisms regulating this switch remain unknown. We have found that a variant of eukaryotic initiation factor (eIF) 4G1 and its homolog, DAP5, factors that are upregulated under stress conditions, bind with high affinity and specificity to the 5’ untranslated regions of these mRNAs and upregulate their translation. These results suggest that binding of eIF4G1 or DAP5 to these mRNAs plays an important role in regulating the switch between the cap-dependent and cap-independent initiation of these mRNAs.

eIF4G1 or DAP5 to these mRNAs plays an important role in regulating the switch between the cap-dependent and cap-independent initiation of these mRNAs. DAP5 bind to oligonucleotides corresponding to the 5' UTRs of a representative set of CITE-mRNAs (CITE-UTRs), namely the HIF-1α, FGF-9, and p53 CITE-UTRs. Complementing these binding assays, we have developed and used a luciferase-based gene expression reporter assay to characterize whether and to what extent binding of ΔN-4G1 and DAP5 promotes the initiation and translation of luciferase-encoding mRNAs containing these same CITE-UTRs, hereafter referred to as CITE-Luc mRNAs, in a rabbit reticulocyte lysate-based in vitro translation system in which cap-dependent initiation has been largely abrogated. The results of our experiments demonstrate that ΔN-4G1 and DAP5 exhibit differential binding affinities to the HIF-1α, FGF-9, and p53 CITE-UTRs that correlate with the differential abilities of these eIFs to drive the initiation and translation of these CITE-Luc mRNAs. Based on our collective results, we propose a model for how CITE-mRNAs regulate the switch from cap-dependent to capindependent initiation and drive expression of the proteins they encode in response to cellular stress.

ΔN-4G1 and DAP5 bind to the CITE-UTRs with affinities that vary among CITE-UTRs and between the two translation factors
To characterize the binding of ΔN-4G1 and DAP5 to the HIF-1α, FGF-9, and p53 CITE-UTRs, we developed a fluorescence anisotropy-based equilibrium binding assay (Fig. 1). This assay reports on the fluorescence anisotropy of a fluorescein fluorophore that is covalently attached to the 5' terminus of the HIF-1α and FGF-9 CITE-UTRs and the two p53 CITE-UTRs corresponding to CITE elements in the 5' UTR and within the coding region, respectively, of the p53 mRNA (hereafter referred to as the p53 A and p53 B CITE-UTRs) and lacking a 5' N7methylguanosine triphosphate cap. In this assay, binding of ΔN-4G1 or DAP5 to the fluoresceinlabeled CITE-UTRs results in an increase in the molecular weight of the CITE-UTRs that is expected to increase the rotational time, and, consequently, the fluorescence anisotropy, of the fluorescein reporter. To quantify the affinity of ΔN-4G1 and DAP5 binding to the four CITE-UTRs, we recorded the change in the fluorescence anisotropy of each fluorescein-labeled CITE-UTR as a function of increasing concentrations of ΔN-4G1 or DAP5 and fitted the resulting data points with a single-site equilibrium binding isotherm ( Fig. 2A and 2B). The results of these experiments, which are reported in Table 1, demonstrate that ΔN-4G1 and DAP5 bind to the four CITE-UTRs with equilibrium dissociation constants (K d s) ranging from 102-300 nM.
Comparative analyses of our results demonstrate that ΔN-4G1 and DAP5 exhibit differential binding affinities both among the CITE-UTRs and between the two translation factors. Specifically, the K d for ΔN-4G1 binding to the p53 B CITE-UTR is more than 2-fold lower than the K d for ΔN-4G1 binding to the p53 A and FGF-9 CITE-UTRs (Table 1). DAP5 exhibited an even greater difference in binding affinity among the CITE-UTRs, with a K d for binding to the FGF-9 CITE-UTR that is more than 2.5-fold lower than the K d for binding to the p53 A CITE-UTR. The difference in binding affinities between the two translation factors was also striking. The K d for DAP5 binding to the FGF-9 CITE-UTR, for example, is almost 2-fold lower than the K d of ΔN-4G1 binding to the same CITE-UTR. Contrasting with this, the K d for ΔN-4G1 binding to the p53 B CITE-UTR is more than 2-fold lower than the K d of DAP5 binding to the same CITE-UTR.

Binding of ΔN-4G1 and DAP5 is specific for particular sequence elements in the CITE-UTRs.
Given previous studies demonstrating that CITE-mRNAs contain highly stable structures in their 5' UTRs (2,9) and proposals that CITE-mRNAs might function by outcompeting non-CITE-mRNAs in the recruitment of limiting amounts of eIFs (15,16) such as eIF4G and DAP5, we conjectured that structural features within the 5' UTRs of the CITE-mRNAs might act as recognition elements and binding sites for ΔN-4G1 and DAP5. Nonetheless, examination of the sequences and predicted secondary structures of the 5' UTRs of the CITE-mRNAs did not lead to identification of any obvious structural features that were common to all four 5' UTRs and that could serve as likely recognition elements and binding sites for ΔN-4G1 or DAP5. We therefore decided to investigate the specificity with which ΔN-4G1 and DAP5 interact with the 5' UTRs of the four CITE-mRNAs. In order to do this, we used our fluorescence anisotropy-based equilibrium binding assay to test the binding of ΔN-4G1 and DAP5 to a presumably unstructured, 101-nucleotide polyUC. The results of these experiments demonstrate that neither ΔN-4G1 or DAP5 exhibit appreciable binding to the polyUC oligonucleotide (Fig. 3), findings consistent with the hypothesis that structural features within the 5' UTRs of the CITE-mRNAs might act as recognition elements and binding sites for ΔN-4G1 and DAP5.
In order to more generally investigate how binding of ΔN-4G1 and DAP5 to RNA depends on structural features within the RNA and to contextualize and validate the results of the binding studies above, we used our fluorescence anisotropy-based equilibrium binding assay to investigate the binding of ΔN-4G1 and DAP5 to a series of RNAs either containing or lacking RNA structural elements that have been previously shown to act as recognition elements and binding sites for eIF4G and/or DAP5. Previously, we have shown that eIF4F binds to the 30-nucleotide iron responsive element (IRE) stem-loop within the 5' UTR of the mRNA encoding ferritin with a K d of 9 nM, a binding interaction that stimulates ferritin mRNA translation in response to elevated cellular concentration of iron (22, 23). More importantly, we showed that eIF4F did not appreciably bind to a control, 30-nucleotide RNA stem-loop, highlighting the specificity with which eIF4F recognizes and binds the IRE. Using our fluorescence anisotropybased equilibrium binding assay, here we show that ΔN-4G1 and DAP5 bind to the IRE with K d s of 18 nM and 35 nM, respectively (Fig. S1). Similarly, we performed experiments in which we quantified the affinity of ΔN-4G1 and DAP5 for the J/K domain of the internal ribosomal entry site (IRES) in the 5' UTR of the mRNA encoding the encephalomyocarditis virus (EMCV) polyprotein ( Fig. S1 and Table S1). These experiments show that ΔN-4G1 and DAP5 bind the EMCV J/K IRES with K d s of 175 nM and 519 nM, respectively, values that are very similar to the K d of 170 nM (24) that has been previously reported for the binding of human eIF4G1(643-1076) to the EMCV J/K IRES as part of the mechanism through which eIF4G1 drives expression of the EMCV polyprotein in human cells. The same report showed that an N-terminal piece of DAP5 (62-330) did not bind to the EMCV J/K IRES, consistent with our much higher K d (519 nM) for full length DAP5 compared to ΔN-4G1 binding. As a further negative control for our CITE-UTR, IRE, and EMCV J/K IRES experiments, we attempted to quantify the affinity of ΔN-4G1 and DAP5 for the uncapped 5' UTR of mRNA encoding human β-actin ( Fig. S1 and Table S1). These control experiments show that ΔN-4G1 and DAP5 do not appreciably bind uncapped βactin mRNA, a result that is consistent with the previous and widespread use of this mRNA as a control for cap dependent initiation and translation (25).

ΔN-4G1 and DAP5 stimulate cap-independent in vitro translation of CITE-Luc mRNAs
In order to determine whether binding of ΔN-4G1 and DAP5 to the 5' UTRs of CITE-mRNAs can stimulate translation of these mRNAs, we developed a luciferase-based gene expression reporter assay and used it to characterize whether and to what extent ΔN-4G1 and DAP5 modulate CITE-mRNA expression. Briefly, the 5' UTRs of the HIF-1α, FGF-9, p53 A , and p53 B CITE-mRNAs and as a control, the 5' UTR of β-actin mRNA were cloned upstream of a luciferase reporter gene and the resulting CITE-Luc mRNAs and β-act-Luc mRNA were translated using a rabbit reticulocyte lysate-based in vitro translation system in which capdependent initiation was suppressed through the addition of a 5' cap analog, m7G(5')ppp(5')G.
Using this assay, we measured the luciferase activity produced by each translation reaction in the absence, as well as in the presence of increasing concentrations, of ΔN-4G1 and DAP5. The results of these experiments show that both ΔN-4G1 and DAP5 can significantly stimulate the cap-independent translation of all four CITE-Luc mRNAs by factors ranging between 2-fold to 5-fold over control reactions performed in the absence of the cap analog or added ΔN-4G1 or DAP5 (Fig. 4). Specifically, ΔN-4G1 stimulates the HIF-1α, FGF-9, p53 A , and p53 B CITE-Luc mRNAs by 5.1, 4.3, 3.6, and 4.6-fold, respectively, and DAP5 stimulates the HIF-1α, FGF-9, p53 A , and p53 B CITE-Luc mRNAs by 3.9, 3.4, 2.6, and 2.3-fold, respectively (Table 2). Notably, the results also show that the concentration of ΔN-4G1 or DAP5 that exhibits the maximum stimulation varies both among the CITE-Luc mRNAs and between the two translation factors. Significantly, neither protein affected the translation of the control β-actin-Luc mRNA ( Fig. S2 and Table 2) indicating the specificity of the stimulation.

Discussion
Using a fluorescence anisotropy-based equilibrium binding assay and a luciferase-based gene expression reporter assay, we have demonstrated that ΔN-4G1 and DAP5 bind to a representative set of CITE-UTRs with Kds in the range of 102-300 nM ( Fig. 2 and Table 1) and stimulate the cap-independent translation of the corresponding CITE-Luc mRNAs by 2-5 fold ( Fig. 4 and Table 2). These Kds are comparable to those observed for the binding of ΔN-4G1 and DAP5 to the IRE stem-loop and the EMCV J/K IRES (Fig.3, Fig. S1, Table 1, and  Table S1), two systems in which it has been previously shown that binding of ΔN-4G1 to a welldefined structural feature within the 5' UTR of an mRNA can stimulates the cap-independent translation of the mRNA (23, 24). Highlighting the specificity of our results, we find that ΔN-4G1 and DAP5 exhibit negligible binding to a polyUC control oligonucleotide, and an uncapped control mRNA encoding β-actin ( Fig. 3, Fig S1, Table 1, and Table S1) and, correspondingly, that ΔN-4G1 and DAP5 do not stimulate the cap-independent translation of the control mRNA encoding β-actin ( Fig. S2 and Table 2). Together with the observation that ΔN-4G1 and DAP5 do not appreciably bind to our control RNAs, the observation that these translation factors bind to the CITE-UTRs studied here with affinities that are similar to those with which they bind the IRE stem-loop and the EMCV J/K IRES strongly suggests that ΔN-4G1 and DAP5 recognize and bind to specific structural features within the CITE-UTRs. Moreover, the observation that ΔN-4G1 and DAP5 exhibit different binding affinities both among the CITE-UTRs and between the two translation factors ( Fig. 4 and Table 2) suggest that ΔN-4G1 and DAP5 differentially recognize different structural features in the various CITE-UTRs. The differential binding affinities of these two proteins for the CITE-UTRs observed here is in agreement with comparative structural studies showing distinct surface properties between the core domains of eIF4G and DAP5 isoforms from human and yeast systems (26).This observation may explain our inability to easily identify a consensus structural feature within the CITE-UTRs studied here that could serve as a likely recognition element and binding site for ΔN-4G1 and DAP5. Thus, the structural basis for ΔN-4G1 and DAP5 recognition and binding to specific sites within CITE-UTRs remains to be determined.
It is likely that the variations in the binding affinities described in the previous paragraph may underlie the variations we observe in the abilities of ΔN-4G1 and DAP5 to stimulate the cap-independent translation of the CITE-mRNAs studied here ( Fig. 2 and Table 1). Indeed, with the exception of the p53B CITE-Luc mRNA, the extent to which ΔN-4G1 stimulates translation of these CITE-Luc mRNAs follows the same trend as that of the Kd for the binding of ΔN-4G1 to the corresponding CITE-UTRs ( Fig. 2 and Table 1). Likewise, with the exception of the p53B CITE-Luc mRNA, the extent to which DAP5 stimulates translation of these CITE-Luc mRNAs follows the same trend as that of the Kd for the binding of DAP5 to the corresponding CITE-UTRs ( Fig. 2 and Table 1). Given the close correlation between these trends, we hypothesize that binding of ΔN-4G1 and DAP5 to CITE-UTRs comprises an important aspect of the mechanism through which these translation factors stimulate the cap-independent translation of CITE-mRNAs. Moreover, the fact that the ΔN-4G1 or DAP5 concentration that exhibits the maximum stimulation of cap-independent translation for the CITE-Luc-mRNAs studied here varies both among the CITE-Luc-mRNAs and between the two translation factors suggests that ΔN-4G1 and DAP5 may play complex roles in the recruitment of the 43S PIC and conversion of the 43S PIC to a 48S PIC.
Our findings are reminiscent of the manner in which viruses, such as EMCV, use highly structured IRESs to directly recruit eIFs, the 40S subunit, and/or the 43S PIC to drive the capindependent translation of viral mRNAs (11,27). Although the mechanisms through which viral mRNAs use IRESs to drive cap-independent initiation have been extensively characterized using genetic, biochemical, and structural approaches (13,28,29), the mechanisms through which eukaryotic cellular mRNAs drive cap-independent initiation remain largely unknown. Nonetheless, an increasing body of evidence suggests eukaryotic cellular mRNAs can employ non-canonical initiation mechanisms that are distinct from those employed by IRES-containing viral mRNAs. For example, Cate and coworkers have recently shown that the 'd' subunit of eIF3 (eIF3d) targets cell proliferation mRNAs and serves as a transcript specific cap-binding protein (30). Using the mRNA encoding the c-Jun transcription factor as an example, these authors showed that, under conditions in which an RNA structure in the 5' UTR blocks eIF4E from binding to the 5' cap, eIF3d binds directly to the 5' cap and serves as an alternative cap binding factor (31). Even more recently, it has been shown that DAP5 is a direct binding partner of eIF3d and it has been proposed that DAP5 used eIF3 to direct the eIF4E-independent cap-dependent translation of a subset of cellular mRNAs when cellular stress conditions lead to inactions of eIF4E (32). In addition to these mechanisms in which viral mRNAs possess IRES elements or eIF3d or other eIFs function as alternative capbinding proteins, the methylation of adenosine residues in the 3'-and 5' UTRs of eukaryotic cellular mRNAs have been shown to stimulate translation by an unknown mechanism (33-35). The CITE-based mechanism we have investigated here is distinct from these other types of mechanisms in that, rather than making use of an alternative cap-binding protein or posttranscriptional modification of the mRNA to be translated, eIF4G1 or DAP5 completely bypass any cap-dependent processes and are instead directly recruited to CITEs within the 5' UTR of CITE-mRNAs. Indeed, because the binding studies we present here employ purified ΔN-4G1 and DAP5 and uncapped RNA oligonucleotides, our results show these translation factors can bind directly to CITE-mRNAs in a completely cap-independent manner and without the need of alternative cap-binding proteins. Similarly, because the gene expression assays we present here also employ purified, uncapped mRNAs and are performed in the presence of a cap analog, we can be confident that the stimulation of translation we observe is due to the direct interaction of ΔN-4G1 and DAP5 with the CITEs in the CITE-mRNAs, rather than to the indirect effects of alternative cap-binding proteins or methylation of the mRNA. Given all of the above, we propose a model for the cap-independent initiation of eukaryotic cellular CITE-mRNAs that bears some similarities to the cap-independent initiation of IREScontaining viral mRNAs (36). Specifically, we propose that eIF4G1 or DAP5, perhaps with the aid of additional factors, bind to structural features within the CITEs of CITE-mRNAs and subsequently recruit additional eIFs, the 40S subunit, and/or the 43S PIC. In analogy to the capindependent initiation of several IRES-containing viral mRNAs (36), we propose that the resulting 48S PIC may then scan to the AUG start codon and potentially establish interactions with RNA structural features and/or RNA-protein complexes at the 3' UTR of the CITE-mRNA that may further enhance expression (29, 37-39). This model provides an attractive mechanism through which CITE-mRNAs can bypass the canonical requirement for eIF4E and undergo capindependent initiation and expression during cellular stress conditions.

Preparation of ΔN-4G1 and DAP5
A plasmid bearing a codon-optimized construct encoding human ΔN-4G1 (encompassing residues 682-1599 of human eIF4G1) (44) which includes the minimal sequence for IRES-mediated cap-independent translation initiation (24) was a generous gift form Dr. Christopher Fraser (University of California at Davis). This construct had an N-terminal, 6x-histidine tag followed by a Flag tag. A tobacco etch virus (TEV) protease cleavage site was introduced after the Flag tag, using Q5 ® Site Directed Mutagenesis Kit from New England Biolabs Inc. The plasmid encoding full length human DAP5 with an N-terminal 6x-histidine tag was purchased from Genscript (Piscataway, NJ). ΔN-4G1 and DAP5 were recombinantly expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells (Agilent) and were purified using a combination of Ni 2+ -nitro-tri-acetic acid (Ni-NTA) affinity and heparin affinity chromatography, as previously described (26, 45). Briefly, the proteins were first purified from bacterial cell lysates using His-Trap HP (Ni-NTA) columns (GE Healthcare life sciences), as per the manufacturer's instructions. The purified 6x-histidine tagged proteins were dialyzed overnight against Storage Buffer (20 mM HEPES pH 7.6, 200 mM KCl, 10 mM β-mercaptoethanol, 10% glycerol) in the presence of TEV protease to cleave off the tags. The untagged proteins were further purified and concentrated using 1 ml HiTrap TM Heparin HP columns (GE Healthcare life sciences). The eluted proteins were analyzed on 10% SDS-PAGE gels and pure fractions (>95% purity) were pooled and dialyzed overnight against Storage Buffer. The concentrations of the purified, concentrated proteins were quantified using Coomassie protein assay reagent (Thermo Scientific) and were aliquoted and stored at -80°C.

Fluorescence anisotropy-based equilibrium binding assays
Fluorescein-labeled RNAs were diluted t0 100 nM using Folding Buffer (20 mM HEPES pH 7.5 and 100 mM KCl) and heated to 90 °C for 2 mins and slowly cooled over 1 hr to room temperature. MgCl 2 to a final concentration of 1 mM was then added to the solution. The solution was then gently mixed and incubated on ice for about 1 hr. Fluorescence anisotropy measurements reporting on the binding of ∆N-4G1 or DAP5 to the fluorescein-labeled RNAs were performed using the equilibrium titration module of an SF-300X stopped-flow fluorimeter (KinTek Corporation, Austin, TX). Fluorescein-labeled RNAs were excited at 495 nm and emission was detected using a 515 nm high-pass filter (Semrock, Rochester, NY). Equilibrium binding titrations began with a 200 µl sample of 100 nM fluorescein-labeled RNA in Titration Buffer (20 mM HEPES, pH 7.6, 100 mM KCl, and 1 mM MgCl 2 ) and 50 data points were collected for each anisotropy measurement by automated continuous injection of 20 µl of ∆N-4G1 or DAP5 over a period of 30 min at a temperature of 25°C. Note that the first injection did not contain any ∆N-4G1 or DAP5. Using the Origin 2018b software package, the data were fitted to a nonlinear, single-site, equilibrium binding equation of the form: where r obs is the observed anisotropy value, r min is the minimum anisotropy value in the absence of ∆N-4G1 or DAP5, r max is the final saturated anisotropy value, [∆N-4G1 or DAP5] is the concentration of ΔN-4G1 of DAP5, and K d is the equilibrium dissociation constant. The chi-squared values (χ 2 ) that represented the statistical goodness of fit were always close to 1 and are reported in Table 1. The equilibrium binding titration of each CITE-UTR was performed three times and fit independently for K d . The fitted K d s were then averaged and the standard deviation calculated ( Table 1).

Preparation of UTR-Luc reporter mRNAs for luciferase-based gene expression reporter assays.
The UTR-Luc mRNA constructs for the luciferase gene expression reporter assays were generated from the BlucB plasmid, (46) which contains a firefly luciferase gene flanked by 5'and 3' UTR sequences of the Barley Yellow Dwarf Virus (BYDV) genomic RNA. To generate each UTR-Luc mRNA reporter construct, a sequences containing the T7 promoter followed by the target CITE-UTR was cloned into the BlucB plasmid vector upstream of the firefly luciferase coding region, after removing the BYDV 5' UTR. Briefly, all UTR-Luc reporter constructs were PCR amplified with NotI forward primer (CTAGGCGGCCGCTAATACGAC) and BssHII reverse primers(HIF1α:CTAGGCGCGCGGTGAATCGGTC, FGF9:CTAGGCGCGCCAGAGGACTCGGC, p53 A : CTAGGCGCGCGGCAGTGAC and p53 B : CTAGGCGCGCTGCTTGGGAC, and β-actin: CTAGGCGC GC GG TGAGCTGG) and then digested with NotI and BssHII. The PCR-amplified sequences were ligated into a NotI-and BssHII-digested BlucB plasmid using DNA ligase (New England Biolabs) and transformed into E. coli DH5-α competent cells. Five colonies were selected, grown overnight in Luria-Bertani (LB) growth media supplemented with 100 µg/mL ampicillin, and used to isolate plasmid DNA using the QIAprep ® Spin Miniprep kit from Qiagen and following the manufacturer's protocol. All clones were confirmed by sequencing (Genewiz).
To generate a linearized plasmid DNA template for in vitro transcription, plasmid DNA was linearized using Kpn1 so as to remove the 3' UTR from the CITE-Luc mRNA reporter construct and the resulting linearized DNA was purified using the GeneJET gel extraction and DNA cleanup Micro Kit from GeneJET and following the manufacurer's instructions. In vitro transcription reactions were preformed using the HiScribe TM T7 Quick High Yield RNA synthesis Kit kit from New England Biolabs Inc and following the manufacturer's protocol. NotI, BssHII, and KpnI were purchased from New England Biolabs and restriction digests were performed according to the manufacturer's instructions.

Luciferase-based gene expression reporter assays
Gene expression was achieved by in vitro translating the UTR-Luc mRNAs using the nucleasetreated rabbit reticulocytes lysate (RRL) in vitro translation system from Promega. Each 25 µl reaction contained 70% v/v of RRL, 0.02 mM amino acid mixture, 0.5 U/µl rRNAsin (Promega), 0.2 mM m7G(5')ppp(5')G cap-analog (Ambion), and varying concentrations of ∆N-4G1 or DAP5 as indicated in the figure legends. Briefly, 100 nM of UTR-Luc mRNA was added to a RRL in vitro translation mixture that had been pre-incubated at 30 °C for 10 mins following the addition of the specified concentration of ∆N-4G1 or DAP5. The resulting in vitro translation reaction was then incubated at 30 °C for 30 mins and stopped by the addition of 60 µM puromycin. Firefly luciferase activity was assayed using a Glomax 96 microplate illuminometer. The achieve this, 3 µl of translation reaction was added to 50 µl Bright-Glo Luciferase assay reagent (Promega) and the resulting luminescence was measured in the illuminometer over a spectral wavelength of 350-650 nm and an integration time of 10 s at room temperature. After subtracting the background measured using an in vitro translation reaction to which no UTR-Luc mRNA had been added, the luminescence data were analyzed and plotted using the Origin 2018b software package. Each UTR-Luc mRNA was translated three times, and the mean and standard deviation of the luminescence data reported. Standard deviation reported is for variation of the mean for data obtained from a single batch of lysate. The mean reported is the value from all batches of lysate. Three to five different rabbit reticulocyte lysates were used for each UTR-Luc mRNA assay. Because of variation in activity among manufacturer's lysate samples, the ∆N-4G1 or DAP5 enhancement varied as much a three-fold.