(A) Schematic of CrPV IGR IRES containing dual-luciferase reporters. Transcription (arrow) is initiated from the PGK1 promoter for yeast-based studies, and from the SV40 early enhancer/promoter for mammalian-based studies. Renilla luciferase is translated by a cap-dependent mechanism, and firefly luciferase synthesis requires cap-independent initiation mediated by the CrPV IGR IRES. (B) CrPV IGR IRES-dependent translation is specifically inhibited in yeast cells expressing the cbf5-D95A mutation. CrPV IGR IRES activity was measured in vivo using a dual luciferase reporter. Activities for cap-dependent and cap-independent translation are normalized to wild-type yeast, graph shows mean ± SEM for n=3. (C) Ribosomes isolated from the cbf5-D95A yeast strain have lower affinities for the CrPV IGR IRES. Filter binding assays were performed using increasing concentrations of radiolabeled CrPV IGR IRES RNA incubated with purified 40S ribosomal subunits from wild-type and cbf5-D95A yeast. A representative graph is shown. The dissociation constants (K_d) ± SEM are indicated. (D) Ribosomes isolated from Dkc1^m MEFs have lower affinities for the CrPV IGR IRES. Filter binding assays were performed using increasing concentrations of radiolabeled CrPV IGR IRES RNA incubated with purified 40S ribosomal subunits from wild-type and Dkc1^m MEF cells. A representative graph is shown. K_ds ± SEM are indicated. (E) 48S pre-initiation complex formation is reduced in Dkc1^m cells. Representative 48S subunit quantification from total cytoplasmic extracts prepared from WT and Dkc1^m cells. Representative profile of a sucrose density gradient showing the radioactive intensity per fraction in WT (black) and Dkc1^m (red) cytoplasmic extracts, respectively (left). (F) Graph shows mean ± SEM of the area under the curve in WT and Dkc1^m cell extracts measured (right). Statistical analysis was carried out using Student t-test. **P<0.01. For all experiments SEM is indicated for n=3. Dkc1^m fibroblasts show significant reductions in 18S rRNA Ψ levels, see also Figure S1.

(A) Schematic of dual-luciferase reporters to monitor translational fidelity. Bicistronic reporters were used to monitor the following: −1 PRF events mediated by signals derived from the yeast L-A virus, HIV-1, or CCR5; +1 PRF promoted by the yeast Ty1 signal; readthrough of a UAA termination codon. In-frame constructs were employed as controls and efficiencies were determined as previously described (Harger and Dinman, 2003; Jacobs and Dinman, 2004). (B) The D95A mutant promotes increased −1 and +1 PRF in yeast. Percent +1 PRF mediated by the Ty1 frameshift signal and −1 PRF promoted by the yeast L-A virus signal were monitored in isogenic wild-type and D95A mutant yeast cells. (C) siRNA knockdown of DKC1 promotes increased −1 PRF in HeLa cells. Percent −1 PRF promoted by signals derived from HIV-1, the human CCR5, or the human IL7-RA mRNA were monitored in HeLa cells transfected with DKC1 siRNA or control. y-axis values represent fold-frameshifting of DKC1 siRNA treated cells compared to cells transfected scrambled siRNAs (control). DKC1 knockdown significantly decreases 18S rRNA Ψ levels, see also Figures S3 and S4. (D) Increased −1 PRF in primary Dkc1^m fibroblasts. Graph shows mean ± SEM of −1 PRF activity promoted by the mouse IL-7RA signal in early passage WT and Dkc1^m fibroblasts. Statistical analysis was carried out using t-test. (E) The D95A mutant promotes increased recognition of a UAA termination codon in yeast. Percent readthrough of a UAA codon was monitored in isogenic wild-type and D95A mutant yeast. Error bars denote standard error of the mean. * P < 0.05. ** P < 0.01.

(A) Single site isotherms of eEF1A stimulated binding of [¹⁴C]Phe-tRNA^Phe (2-fold serial dilutions from 1 to 128 pmoles) to the A-sites of 20 pmoles of poly(U) primed ribosomes pre-loaded with tRNA^Phe in their A-sites. (B) Single site isotherms of Ac-[¹⁴C]Phe-tRNA^Phe (2-fold serial dilutions from 1 to 128 pmoles) to P-sites of 20 pmoles of poly(U) primed ribosomes. These studies were carried out as previously described (Meskauskas and Dinman, 2010). Steady-state K_d values and standard deviations for each sample are indicated. All tRNA binding assays were performed in triplicate. (C) Ten-fold dilution spot assays of isogenic wild-type and D95A mutants in the presence of paromomycin (left panel) and anisomycin (center panel). Filter disc assays were used to score changes in sensitivity to sparsomycin (right panels). D95A cells showed similar sensitivity to high and low temperatures, see also Figure S2.