Supporting Figures

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Fig. 5. Insertion of a strong 5' hairpin structure optimizes monitoring of bona fide internal ribosome entry sequence (IRES) activity. HeLa cell extracts were programmed with ApppG-capped luciferase reporter mRNAs with or without a stable hairpin upstream of a spacer sequence and the IRES, as indicated. Reporter mRNAs lacking the c-myc IRES upstream of the firefly luciferase ORF were used as negative controls. IRES activity is defined as the difference in activity above that of the corresponding construct lacking the IRES. Error bars denote the standard deviation from the mean of at least three independent experiments.

Fig. 6. Concentrations of eukaryotic translation initiation factor (eIF) 4GI and p97 in HeLa cell-free translation extracts. (A) Western blot analysis of endogenous eIF4GI in HeLa cell extracts. HeLa cell extracts were pretreated with coxsackieviral protease 2A, resulting in a complete cleavage of eIF4GI. The C-terminal cleavage product of eIF4GI was compared with recombinant mc4G. (B) Comparative Western blot analysis of endogenous p97 in HeLa cell extracts and recombinant His-tagged p97.

Fig. 7. Addition of eIF4F rescues cap- as well as c-myc IRES-dependent translation in eIF4GI-depleted HeLa cell extracts. Mock- or eIF4GI-depleted HeLa cell extracts were programmed with the indicated reporter mRNAs in the presence of buffer (-)or 4 pmol of eIF4F purified from reticulocyte lysate. Error bars denote the standard deviation from the mean of three independent experiments.

Fig. 8. Addition of m4G or p97 mediates c-myc IRES-driven translation of a bicistronic reporter in eIF4GI-depleted HeLa cell extracts. A schematic representation of the bicistronic c-myc IRES-containing reporter mRNA is shown above the bar graphs. Mock- or eIF4GI-depleted HeLa cell extracts were programmed with the reporter mRNA in the presence of dialysis buffer (-), 6 pmol of m4G, or 5 pmol of p97, as indicated. Firefly luciferase activity is plotted on the y axis. Translation in mock-depleted HeLa cell extracts was defined as 100%. Error bars denote the standard deviation from the mean of three independent experiments.

Fig. 9. The middle domain of eIF4GI or p97 also stimulates translation driven by pro- and anti-apoptotic IRESs in mock-depleted HeLa cell extracts. Mock- or eIF4GI-depleted HeLa cell extracts were programmed with the indicated mRNAs in the presence of dialysis buffer (-), 6 pmol of m4G (A), or 5 pmol of p97 (B). Translation of the respective mRNAs after addition of dialysis buffer was defined as 100%. Error bars denote the standard deviation from the mean of at least three independent experiments.

Fig. 10. Depletion of eIF4GI or addition of m4G or p97 does not alter reporter mRNA stability in HeLa cell extracts. Mock- or eIF4GI-depleted HeLa cell extracts were programmed with [³²P]UTP-labeled reporter mRNAs in the presence of dialysis buffer (-), 6 pmol of m4G, or 5 pmol of p97, as indicated. After completion of the in vitro translation reaction, [³²P]UTP-labeled RNA was added as a recovery control, and total RNA was isolated. Following separation on an agarose gel, reporter mRNA levels were assessed using autoradiography and normalization to the levels of control RNA. Reporter mRNA levels in mock-depleted HeLa cell extracts were defined as 100%.

Fig. 11. m4G and p97 also mediate c-myc IRES-driven translation of a reporter lacking the 200-nt spacer region between the hairpin and IRES in eIF4GI-depleted HeLa cell extracts. A schematic representation of the c-myc IRES-containing reporter mRNA is shown above the bar graphs. Mock- or eIF4GI-depleted HeLa cell extracts were programmed with the reporter mRNA in the presence of dialysis buffer (-), 6 pmol of m4G, or 5 pmol of p97, as indicated. Translation in mock-depleted HeLa cell extracts was defined as 100%. Error bars denote the standard deviation from the mean of three independent experiments.

Fig. 12. The middle fragment of eIF4GII fails to mediate c-myc IRES-driven translation in eIF4GI-depleted HeLa cell extracts. (A) Comparative Western blot analysis of endogenous eIF4GII in HeLa cell extracts and recombinant His-tagged middle fragment of eIF4GII (m4GII). (B) Mock- or eIF4GI-depleted HeLa cell extracts were programmed with the indicated c-myc IRES reporter mRNA in the presence of dialysis buffer (-), 6 pmol of m4GI, or 6 pmol of m4GII. Translation in mock-depleted HeLa cell extracts after addition of dialysis buffer was defined as 100%. Error bars denote the standard deviation from the mean of at least three independent experiments.