1. Volume 17, Issue 3, Pages 405-416 (February 2005)
Ribosome Stalling Regulates IRES-Mediated Translation in Eukaryotes, a Parallel to Prokaryotic Attenuation 
James Fernandez, Ibrahim Yaman, Charles Huang, Haiyan Liu, Alex B. Lopez, Anton A. Komar, Mark G. Caprara, William C. Merrick, Martin D. Snider, Randal J. Kaufman, Wouter H. Lamers, Maria Hatzoglou 
Molecular Cell 
Volume 17, Issue 3, Pages (February 2005)
DOI: /j.molcel

2. Figure 1
Cx Induces cat-1 IRES-Mediated Translation in a Manner that Requires Interactions within the RNA Leader
(A and D) Schematic representation of mRNAs transcribed from the bicistronic (A) and monocistronic (D) expression vectors. Bicistronic vectors contain chloramphenicol acetyltransferase (CAT) as the first and firefly luciferase (LUC) as the second cistron. Monocistronic vectors contain only the LUC cistron. Sequences from the cat-1 UTR were introduced into the intercistronic region (A) or 5′-UTR (D) (indicated by the dotted lines). A detailed description of the cat-1 5′-UTR was previously published (Fernandez et al., 2002a). Construct M4 (D) contains the cat-1 gene promoter and the entire 270 nt UTR (Fernandez et al., 2003).
(B and C) C6 rat glioma cells were transiently transfected with B1 (B) or the indicated constructs (C).
(E) Monocistronic expression vectors were cotransfected with a β-galactosidase-expressing plasmid.
In all experiments, 48 hr after transfection, cells were incubated with or without the indicated concentrations of Cx (B) or 50 μg/ml Cx (C and E). Cell lysates were prepared and analyzed for LUC, CAT, and β-galactosidase activities at the indicated times (B) or 4 hr (C and E). Activity/μg protein values were normalized to the values for B1 in cells without Cx (B and C). Values for monocistronic mRNAs are expressed as LUC/βGal (E). Values are mean ± standard error of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Cx Induction of Translation from the cat-1 IRES Requires Translation Initiation at the Downstream AUG Initiation Codon and Is Independent of eIF2α Phosphorylation
C6 (A), MEF S/S, and MEF A/A (B) cells were transiently transfected with the indicated vectors. After transfection (48 hr), cells were incubated with or without 50 μg/ml Cx for 4 hr. Cell lysates were prepared and analyzed for LUC and CAT activities. The LUC/CAT values were normalized to the value for B1 in cells without Cx. Values are mean ± standard error of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
The Position of the cat-1 uORF Stop Codon Is Important for Cx-Induced cat-1 IRES-Mediated Translation
(A) C6 cells were transfected with either the B1 bicistronic mRNA expression vector (stop codon at position −80) or mutants with stop codons at positions −209 to −11 (D). All mutants had U−80GA mutagenized to U−80UA. Constructs were transfected into C6 cells, and the effects of Cx were analyzed as in Figure 2. The LUC/CAT values were normalized to untreated cells transfected with B1. Values are mean ± standard error of three independent experiments. (B–D) Nuclease and chemical sensitivity mapping of an RNA containing nucleotides −270 to +1 of the cat-1 leader was performed as described in the Experimental Procedures using RNase T1 (T1), RNases T1 and A (T1/A), RNase V1 (V1), or DEPC/aniline (DEPC). The table in (B) shows a summary of cleavages of the region A−224 to A−6. A model of nucleotides −224 to +1 of the cat-1 mRNA leader with the cleavage sites is shown in (C). (D) shows the position of the stop codon mutants used in (A).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Introduction of Rare Codons into the cat-1 uORF Enhances Basal Activity of the cat-1 IRES in a Manner Dependent on uORF Translation
(A) Schematic representation of mRNAs transcribed from bicistronic expression vectors B1 and B19–B22.
(B) Vectors were transfected into C6 cells, and the effect of the indicated concentrations of Cx were determined as described in Figure 2. The LUC/CAT ratios were normalized to the B1 control value.
(C) Effect of Cx on the incorporation of radioactivity by C6 cells incubated with 35S-Met for 2 hr. Incorporation of 35S-Met, expressed as percent of Cx-free control, is plotted against the Cx concentration. Mean ± standard error from triplicate determinations is shown.
(D) C6 cells were incubated in 50 μg/ml Cx for the indicated times, incubated in Met-free medium for 15 min, and then labeled with 35S-Met for 15 min in the presence of Cx. Mean ± standard error from triplicate determinations is shown.
(E) Vectors were transfected into C6 cells, and the effects of Cx (4 hr, 50 μg/ml) or amino acid starvation (9 hr) were determined. The LUC/CAT ratios are shown.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Introduction of Rare Codons into the cat-1 uORF Modulates cat-1 IRES-Mediated Translation In Vitro
(A) Capped bicistronic RNAs were translated in RRL as described in the Experimental Procedures. LUC and CAT activities were measured and normalized to the values for B1 RNA.
(B) RNAs were translated in RRL in the presence of 35S-Met, and labeled products were analyzed on an SDS-PAGE gel. The positions of LUC and CAT proteins (arrows) and globin (*) are shown. The bands between the CAT and LUC proteins are aborted translation products because they were not seen when RNA was omitted (data not shown). The two lanes on the right are an overexposed sample to clearly show the uORF peptide.
(C) The indicated mono- and bicistronic RNAs were translated in RRL. LUC activities from triplicate determinations (mean ± standard error) are shown. The graph shows results with the constructs on the right from top (1) to bottom (4).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
A Model of Ribosome Stalling as a Regulator of Translation Initiation in Eukaryotes and Prokaryotes
Translational control for two mRNAs with small uORFs are shown: (1) In prokaryotes, ermC methylase mRNA translation is regulated by structural remodeling of the leader that hides or exposes the methylase SD sequence and initiator AUG. Erythromycin-induced ribosome stalling within the 19 residue leader peptide exposes these sequences, promoting methylase translation. (2) In eukaryotes, cat-1 arginine/lysine transporter mRNA translation is regulated by structural remodeling that mediates formation of an active IRES. This remodeling is promoted by ribosome stalling within the uORF of the mRNA leader.
Molecular Cell  , DOI: ( /j.molcel )