1. Volume 15, Issue 6, Pages 925-935 (September 2004)
Enhancement of IRES-Mediated Translation of the c-myc and BiP mRNAs by the Poly(A) Tail Is Independent of Intact eIF4G and PABP 
Christian Thoma, Giovanna Bergamini, Bruno Galy, Patrick Hundsdoerfer, Matthias W. Hentze 
Molecular Cell 
Volume 15, Issue 6, Pages (September 2004)
DOI: /j.molcel

2. Figure 1
The Poly(A) Tail Enhances Translation Driven from the c-myc and BiP IRESs In Vitro
(A) A schematic representation of the IRES-containing reporter mRNAs is shown above the bar graphs. HeLa cell extracts were programmed with 50 ng ApppG-capped c-myc (left panel) or BiP (right panel) IRES reporter mRNAs. The respective inverted IRESs were used as negative controls. Luciferase activity is expressed as relative light units. Error bars denote the standard deviation from the mean of three different experiments. The physical stability of the mRNAs was assessed by Northern blotting. CAT RNA was added to each sample as a recovery control.
(B) Stimulation by the poly(A) tail [in multiples of the value from corresponding controls lacking a poly(A) tail] of cap-dependent and IRES-dependent translation in buffer (100%) or micrococcal nuclease-treated HeLa cell extracts (ranging from to 0.04 U micrococcal nuclease per translation reaction). The data represent averages of three independent determinations. The effectiveness of the nuclease treatment in removing endogenous mRNAs was assessed by 35S-Met incorporation and SDS-PAGE followed by autoradiography. In (A) and (B), the concentration of KOAc was 80 mM for the c-myc and BiP IRESs and 40 mM for capped mRNAs.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
The Poly(A) Tail Stimulates Translation Driven by the c-myc IRES In Vivo
(A) HeLa cells were transfected for 1 hr with the indicated monocistronic mRNAs and subsequently washed (0 hr). At 0 hr, luciferase activity was barely above background (data not shown). 6 hr after transfection, luciferase activity was measured and expressed as relative light units (the data show the average of three experiments with the standard deviations from the mean). Total RNA was isolated at the indicated times, and the integrity of firefly luciferase mRNA was analyzed by Northern blotting. The ribosomal 28S and 18S rRNAs on the ethidium bromide-stained gel used for Northern blotting are shown as controls.
(B) HeLa cells were transfected with bicistronic mRNAs either containing a stable stem-loop structure (SL) in the 5′ UTR or not. In vivo assay and luciferase activity analysis was performed as described in (A). 6 hr after transfection, total RNA was isolated and the integrity of the reporter mRNAs was analyzed by Northern blotting. GAPDH was used as control. Note that the IRES-mediated translation of the second cistron is stimulated in the presence of a (5′) stem loop insertion.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Translation from the c-myc and BiP IRESs Requires eIF4A
(A) The indicated monocistronic mRNAs were translated in nuclease-treated extracts. RNAs driven by the c-myc or BiP IRESs contained a cap analog ApppG. Dialysis buffer (−), wild-type eIF4A (w, 2 μg) or a dominant-negative mutant of eIF4A (m, 2 μg) were added to the translation reactions. The data represent the average of three experiments with error bars indicating standard deviations from the mean.
(B) Translation reactions were programmed with the indicated mRNAs in the presence of dialysis buffer (−), wild-type eIF4A (w, 0.5, 1.0, 2.0, and 4.0 μg, respectively) or a dominant-negative mutant of eIF4A (m, 2 μg) and analyzed as in (A). The stimulation by the poly(A) tail was 9.0-, 4.0-, 8.0-, 4.9-, and 12.4-fold for cap-dependent, EMCV-, PV-, c-myc-, and BiP IRES-driven translation, respectively, without addition of eIF4A. In (A) and (B), the concentration of KOAc was 100 mM for the polio, EMCV, c-myc, and BiP IRESs, 145 mM for the HCV IRES, and 60 mM for capped mRNAs.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Role of eIF4G in Translation from the c-myc and BiP IRESs and Its Enhancement by the Poly(A) Tail
(A) Schematic representation of eIF4G with protein binding sites and the protease 2A cleavage site indicated by arrows (left panel). Western blot analysis of eIF4GI, eIF4GII, and PABP in control and protease 2A-treated extracts is shown on the right. The positions of intact eIF4GI, eIF4GII, PABP, and the respective cleavage products are indicated by arrows. The antibodies detecting eIF4GI and eIF4GII were raised against the C-terminal part of the respective protein. Asterisks denote crossreactive nonspecific bands.
(B) Nuclease-treated extracts were preincubated with coxackievirus 4B protease 2A to cleave eIF4GI and eIF4GII to completion, or with control buffer (40 mM HEPES, 10% glycerol). Following protease treatment, the translation reactions were programmed with the indicated mRNAs.
(C) Translation reactions were programmed with bicistronic mRNAs and analyzed as in (B).
(D) The monocistronic mRNAs were translated following addition of dialysis buffer (−) or a recombinant fragment representing the C-terminal two-thirds of eIF4GI (aa 486–1404) (C-4G, 1 μg). In (B)–(D), the data represent the average of three experiments with error bars indicating standard deviations from the mean. The concentration of KOAc was 100 mM for the polio and EMCV IRESs, 80 mM for the c-myc and BiP IRESs, and 40 mM for capped mRNAs.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 The Poly(A) Tail Stimulates c-myc mRNA Translation In Vitro
Nuclease-treated extracts were preincubated with coxackievirus 4B protease 2A as described in Figure 4. ApppG-capped c-myc mRNA with or without a poly(A) tail was translated as described previously (Bergamini et al., 2000), with the following modifications: for labeling of c-myc proteins, [35S] methionine and cysteine (1.43 mCi/ml) were substituted for unlabeled methionine and cysteine in the translation mix containing 100 μM amino acids, 150 μM spermidine, 1.3 mM MgOAc, and 70 mM KOAc. Translation products were immunoprecipitated. The position of c-myc proteins are indicated. The asterisk denotes a nonspecific band also seen in samples without added RNA.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Role of PABP in IRES-Mediated Translation
(A) The indicated mRNAs were translated with 84 mM (c-myc and BiP IRES) or 44 mM (capped mRNAs) KOAc in nuclease-treated extracts in the absence (−) or presence (+) of exogenously added recombinant PAIP2 (32 μg/ml). The stimulation by the poly(A) tail compared to the corresponding mRNA lacking a poly(A) tail is expressed as multiples of the mRNAs lacking a poly(A) tail. The data represent the average of three experiments with error bars indicating standard deviations from the mean.
(B) Western blot analysis of eIF4GI, eIF4GII, PABP, eIF4A, and actin in non-, mock- or PABP-depleted extracts. An overexposure of the blot probed with mouse monoclonal anti-PABP antibody is shown on the right to reveal residual PABP (5%–10%) in the depleted extracts.
(C) The indicated mRNAs were translated with 82 mM (BiP IRES) or 42 mM (capped mRNAs) KOAc in the absence (−) or presence (+) of recombinant PABP (10 μg/ml). The stimulation by the poly(A) tail compared to the corresponding mRNAs lacking a poly(A) tail is expressed as multiples of the (A−) mRNAs. The data represent the average of three experiments with error bars indicating standard deviations from the mean.
Molecular Cell  , DOI: ( /j.molcel )