An mRNA-specific tRNAi carrier eIF2A plays a pivotal role in cell proliferation under stress conditions: Stress-resistant translation of c-Src mRNA is mediated by eIF2A

c-Src, a non-receptor protein tyrosine kinase, activates NF-��B and STAT3, which in turn triggers the transcription of anti-apoptosis- and cell cycle-related genes. c-Src protein regulates cell proliferation, cell motility and programmed cell death. And the elevated level of activated c-Src protein is related with solid tumor generation. Translation of c-Src mRNA is directed by an IRES element which mediates persistent translation under stress conditions when translation of most mRNAs is inhibited by a phosphorylation of the alpha subunit of eIF2 carrying the initiator tRNA (tRNAi) to 40S ribosomal subunit under normal conditions. The molecular basis of the stress-resistant translation of c-Src mRNA remained to be elucidated. Here, we report that eIF2A, an alternative tRNAi carrier, is responsible for the stress-resistant translation of c-Src mRNA. eIF2A facilitates tRNAi loading onto the 40S ribosomal subunit in a c-Src mRNAdependent manner. And a direct interaction between eIF2A and a stem-loop structure (SL I) in the c-Src IRES is required for the c-Src IRES-dependent translation under stress conditions but not under normal conditions. Finally, we showed that the eIF2Adependent translation of c-Src mRNA plays a pivotal role in cell proliferation under stress conditions. ? The Author(s) 2016.115sciescopu

The bent conformation of poly(A)-binding protein induced by RNA-binding is required for its translational activation function

A recent study revealed that poly(A)-binding protein (PABP) bound to poly(A) RNA exhibits a sharply bent configuration at the linker region between RNA-recognition motif 2 (RRM2) and RRM3, whereas free PABP exhibits a highly flexible linear configuration. However, the physiological role of the bent structure of mRNA-bound PABP remains unknown. We investigated a role of the bent structure of PABP by constructing a PABP variant that fails to form the poly(A)-dependent bent structure but maintains its poly (A)-binding activity. We found that the bent structure of PABP/poly(A) complex is required for PABP's efficient interaction with eIF4G and eIF4G/eIF4E complex. Moreover, the mutant PABP had compromised translation activation function and failed to augment the formation of 80S translation initiation complex in an in vitro translation system. These results suggest that the bent conformation of PABP, which is induced by the interaction with 30 poly(A) tail, mediates poly(A)-dependent translation by facilitating the interaction with eIF4G and the eIF4G/eIF4E complex. The preferential binding of the eIF4G/eIF4E complex to the bent PABP/poly(A) complex seems to be a mechanism discriminating the mRNA-bound PABPs participating in translation from the idling mRNA-unbound PABPs.111Ysciescopu

CDK11/p58 represses general translation during mitosis through eIF3F phosphorylations

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eIF4GI plays a role in the miRNA-mediated gene silencing of the poly(A)-tailed m⁷G-capped mRNA.

<p>(<i>A</i>) Schematic diagrams of the reporter mRNAs used here. The abbreviations of each reporter are depicted in parentheses at the left side. (<i>B</i>) The effect of the 5′ cap structure and the poly(A) tail on the miRNA-mediated translational repression. m⁷G-capped/A-capped and/or poly(A)-tailed/nonadenylated FL mRNAs were co-transfected with the nonadenylated m⁷G-capped RL mRNAs in the presence of miControl or miCXCR4 into HeLa cells. After 19 h of incubation, the luciferase activities were measured to obtain the FL/RL activity ratios. (<i>C</i>) Total RNAs from the cells in panel B were extracted, and the relative levels of the mRNAs were analyzed by quantitative RT-PCR analysis. In panels <i>B</i> and <i>C</i>, the values from miControl-treated cells in all experiments were represented as 100% (lane 1). (<i>D</i>) De-repression of the miRNA-mediated gene silencing by siRNAs against translation factors using the RNA reporter system. The overall procedures for experiments and analyses were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#s2" target="_blank">Materials and Methods</a>. All experiments were performed in triplicate. In panels <i>B</i> and <i>D</i>, the P-values are described (Student <i>t</i>-test). Standard deviations are indicated by bars.</p

eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing

<div><p>MicroRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene silencing by binding to complementary target mRNAs and recruiting the miRNA-containing ribonucleoprotein complexes to the mRNAs. However, the molecular basis of this silencing is unclear. Here, we show that human Ago2 associates with the cap-binding protein complex and this association is mediated by human eIF4GI, a scaffold protein required for the translation initiation. Using a cap photo-crosslinking method, we show that Ago2 closely associates with the cap structure. Taken together, these data suggest that eIF4GI participates in the miRNA-mediated post-transcriptional gene silencing by promoting the association of Ago2 with the cap-binding complex.</p> </div

eIF4GI mediates the Ago2-cap association.

<p>(<i>A</i>) Identification of a translation initiation factor capable of augmenting the Ago2-cap association. WCEs from 293FT cells co-transfected with plasmids expressing myc-Ago2 and Flag-tagged translation factors (eIF3c, eIF4AI, eIF4E, eIF4GI and PABP) were subjected to cap-pulldown assays. The expression levels of the transfected genes (lanes 1–6), their cap-associations (lanes 7–12), and various proteins from WCEs or from the resin-bound fractions were monitored by Western blotting with the indicated antibodies. (<i>B</i>) The eIF4GI-dependent cap-association of Ago2. WCE from HeLa cells transfected with si-Control or si-eIF4GI were applied to cap-pulldown assays. The knock-down efficiency of siRNAs and the resin-bound proteins were monitored using antibodies described. (<i>C</i>) Determination of a domain in eIF4GI responsible for augmenting the Ago2-cap association. WCEs from 293FT cells transiently expressing myc-Ago2 and Flag-tagged fragments encoding the N-terminal, middle or C-terminal regions of eIF4GI (depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#pone-0055725-g005" target="_blank">Figure 5A</a>) were subjected to cap-pulldown assays. The resin-bound Ago2 (upper panel), the ectopically expressed Ago2 and eIF4GI fragments (lower panel), GAPDH and eIF4E proteins were detected using the indicated antibodies.</p

The 5′ cap structure may directly contact with Ago2.

<p>(<i>A</i>) WCEs (2 mg) from 293FT cells transfected with plasmids expressing Flag-Ago1, Flag-Ago2, Flag-Dicer, Flag-TNRC6C or Flag-eIF4E were subjected to Flag-IP, and cap-labeled RNAs were then incubated with the immunoprecipitated proteins in the presence or absence of cap analogs. The RNAs and proteins were photo-crosslinked by UV irradiation and then analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#s2" target="_blank">Materials and methods</a> (upper panel). In parallel, Western blot analyses with an anti-Flag antibody were used to measure the amounts of the precipitated Flag-tagged proteins (lower panel). The positions of the proteins on the gel are denoted by arrows. (<i>B</i>) The effect of eIF4GI on the cap-crosslinking of Ago2. The resin-bound proteins from Flag-IP experiments using 293FT cell extracts expressing Flag-Ago2 and myc-eIF4GI were subjected to a cap-crosslinking assay (upper panel) and Western blotting (lower panel). (<i>C</i>) The effect of the N-terminal part of eIF4GI on the cap-crosslinking of Ago2. 293FT cells were co-transfected with plasmids expressing Flag-Ago2 and myc-tagged eIF4GI-N (aa 42–622), and a cap-crosslinking assay was performed.</p

The intact Ago2 associates with the cap-binding complex.

<p>(<i>A</i>) A schematic diagram of human Ago2 and the constructs used for co-immunoprecipitation and cap-pulldown assays. (<i>B</i>) The domains in Ago2 required for the association with eIF4GI. 293FT cells were transfected with plasmids expressing Flag-tagged N-, M- or C-terminal portions of eIF4GI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#pone-0055725-g005" target="_blank">Figure 5A</a>) together with plasmids expressing myc-tagged Ago2 fragments corresponding to the N, PAZ/M or PIWI domains. Extracts from the transfected cells were incubated with the Flag-resin and the precipitated proteins were visualized by Western blotting using the indicated antibodies. (<i>C</i>) RNA-independent association of Ago2 domains with eIF4GI domains. RNase-treated (lanes 2, 4, 6, 8, 10 and 12) or -untreated (lanes 1, 3, 5, 7, 9 and 11) WCE from 293FT cells transiently expressing Flag-tagged eIF4GI derivatives (eIF4GI-N, -M or -C) with myc-tagged Ago2 variants (Ago2-N, -PAZ/M or -PIWI) were subjected to the immunoprecipitation experiments using the Flag-resins. The bound proteins were detected using antibodies indicated. (<i>D</i>) Association of Ago2 domains with the cap-resin. 2 mg of WCEs from 293FT cells transfected with plasmids expressing myc-tagged Ago2 derivatives were applied to cap-pulldown assays and the bound proteins were monitored using anti-myc antibodies. For comparison, various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5.</p

RACK1 facilitates HCV preliferation by inducing autophagy of host cells

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Human Ago associates with the cap-binding complex.

<p>(<i>A</i>) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. (<i>B</i>) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. (<i>C</i>) The cap-associations of ectopically expressed proteins were monitored as in panel <i>A</i>, except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. (<i>D</i>) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m⁷G(5′)ppp(3′)G (lane 4). (<i>E</i>) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels <i>A</i> and <i>C</i>, various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels <i>A</i>, <i>C</i>, <i>D</i> and <i>E</i>, Western blot analyses were performed using the indicated antibodies.</p