Volume 44, Issue 4, Pages 660-666 (November 2011) rRNA Pseudouridylation Defects Affect Ribosomal Ligand Binding and Translational Fidelity from Yeast to Human Cells  Karen Jack, Cristian Bellodi, Dori M. Landry, Rachel O. Niederer, Arturas Meskauskas, Sharmishtha Musalgaonkar, Noam Kopmar, Olya Krasnykh, Alison M. Dean, Sunnie R. Thompson, Davide Ruggero, Jonathan D. Dinman  Molecular Cell  Volume 44, Issue 4, Pages 660-666 (November 2011) DOI: 10.1016/j.molcel.2011.09.017 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 660-666DOI: (10.1016/j.molcel.2011.09.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 rRNA Pseudouridylation Defects Inhibit IRES-Dependent Translation in Yeast and Mammalian Cells through a Defect in Ribosome Binding to IRES Elements (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 WT 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 WT and cbf5-D95A yeast. A representative graph is shown. The dissociation constants (Kd) ± SEM are indicated. (D) Ribosomes isolated from Dkc1m 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 WT and Dkc1m MEF cells. A representative graph is shown. Kds ± SEM are indicated. (E) 48S preinitiation complex formation is reduced in Dkc1m cells. Representative 48S subunit quantification from total cytoplasmic extracts prepared from WT and Dkc1m cells. Representative profile of a sucrose density gradient showing the radioactive intensity per fraction in WT (black) and Dkc1m (light gray) cytoplasmic extracts, respectively (left). (F) Graph shows mean ± SEM of the area under the curve in WT and Dkc1m cell extracts measured (right). Statistical analysis was carried out using Student's t test. ∗∗p < 0.01. For all experiments, SEM is indicated for n = 3. Dkc1m fibroblasts show significant reductions in 18S rRNA Ψ levels, see also Figure S1. Molecular Cell 2011 44, 660-666DOI: (10.1016/j.molcel.2011.09.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 rRNA Pseudouridylation Defects Affect Translational Fidelity (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 WT and D95A mutant yeast cells. (C) siRNA-mediated DKC1 downregulation significantly reduces total rRNA pseudouridylation levels in human cells. HeLA cells were transduced with scramble control or DKC1 siRNA constructs. Total 18S (left) and 28S (right) rRNA pseudouridylation levels were isolated 48 hr after transfection and analyzed using HPLC. Graphs show mean ratio Ψ/C ± SEM of four independent experiments performed in triplicate. (D) 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 was 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 with scrambled siRNAs (control). DKC1 knockdown significantly decreases 18S rRNA Ψ levels, see also Figure S3. (E) Increased −1 PRF in primary Dkc1m fibroblasts. Graph shows mean ± SEM of −1 PRF activity promoted by the mouse IL-7RA signal in early passage WT and Dkc1m fibroblasts. (F) The D95A mutant promotes increased recognition of a UAA termination codon in yeast. Percent readthrough of a UAA codon was monitored in isogenic WT and D95A mutant yeast. Error bars denote standard error of the mean. Statistical analysis was carried out using Student's t test. ∗p < 0.05. ∗∗p < 0.01. Molecular Cell 2011 44, 660-666DOI: (10.1016/j.molcel.2011.09.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Global Ψ Defects Affect the Sensitivity of Yeast to Translational Inhibitors by Promoting Decreased Ribosomal Affinities for tRNAs in the A and P Sites (A) Single-site isotherms of eEF1A stimulated binding of [14C]Phe-tRNAPhe (2-fold serial dilutions from 1 to 128 pmoles) to the A sites of 20 pmoles of poly(U) primed ribosomes preloaded with tRNAPhe in their A sites. (B) Single-site isotherms of Ac-[14C]Phe-tRNAPhe (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 Kd 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 WT 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. Molecular Cell 2011 44, 660-666DOI: (10.1016/j.molcel.2011.09.017) Copyright © 2011 Elsevier Inc. Terms and Conditions