SiRNA-mediated silencing of <i>RPL12</i> increases ΔF508-CFTR expression, PM density and function at 37°C.

<p><b>(A)</b> PM density of WT and ΔF508-CFTR upon <i>RPL12</i> knockdown in CFBE at 37°C (<i>n</i> = 3). <b>(B)</b> Effect of <i>RPL12</i> knockdown on the expression pattern of ΔF508-CFTR determined by immunoblotting in CFBE (upper panel). CFTR was visualized using anti-HA antibody, anti-Na⁺/K⁺-ATPase served as loading control. Densitometric analysis (lower panel, <i>n</i> = 3) of the core-glycosylated (band B, filled arrowhead) ΔF508-CFTR is expressed as percent of controls transfected with NT siRNA (<i>n</i> = 3). <b>(C)</b> The effect of <i>RPL12</i> knockdown, VX-809 treatment (3 μM, 24 h) or combination of both on the expression pattern of ΔF508-CFTR expressed constitutively in CFBE cells (CFBE^c). Immunoblots (upper panel) were probed with antibodies against CFTR (10B6.2), Rpl12, and β-actin as a loading control. Expression levels of Rpl12 and the core- (band B, filled arrowhead) or complex-glycosylated (band C, empty arrowhead) ΔF508-CFTR were quantified by densitometry and are expressed as a percentage compared to controls transfected with NT siRNA (lower panel, n = 3). <b>(D)</b> <i>RPL12</i> knockdown increases the function of ΔF508-CFTR at physiologic temperature as determined by halide sensitive YFP quenching assay (<i>n</i> = 3). <b>(E)</b> Representative short-circuit current (I_sc) recordings (upper panel) and quantification of the changes (ΔI_sc, <i>n</i> = 5, lower panel) in CFBE^c monolayers expressing ΔF508-CFTR after siRNA-mediated <i>RPL12</i> knockdown or NT siRNA transfection. CFTR mediated currents were induced by sequential acute addition of Frk (10 μM) and gen (50 μM) followed by CFTR inhibition with inhibitor₁₇₂ (10 μM) in the presence of a basolateral-to-apical chloride gradient after basolateral permeabilization with amphotericin B (100 μM). The values in D and E were normalized to account for the increase in ΔF508-CFTR mRNA upon siRPL12_6 treatment. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–5 independent experiments. The underlying data of panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

<i>RPL12</i> knockdown increases the maturation efficiency of some misfolded membrane proteins.

<p><b>(A–D)</b> PM densities of the indicated proteins were measured by cell surface ELISA in NT or <i>RPL12</i> siRNA transfected HeLa cells. In A–C, cells stably expressing extracellular HA-epitope tagged V2R-Y128S (A, <i>n</i> = 3), MLC1-P92S and S280L (B, <i>n</i> = 3) or hERG-WT and G601S (C, <i>n</i> = 4) were used. The endogenous TfR was labeled with biotin-Tf and detected with neutravidin-HRP (D, <i>n</i> = 3). SiRNAs for CHIP and Tsg101 served as positive controls that attenuated degradation of misfolded PM proteins from post-Golgi compartments. <b>(E)</b> The maturation efficiency of hERG-G601S was determined by metabolic pulse chase experiment using ³⁵S-methinonine and ³⁵S-cysteine in HeLa cells transfected with <i>RPL12</i> or NT siRNAs. Pulse labeling was performed for 20 min at 26°C, followed by 3 h chase at 37°C. The maturation efficiency of the channel was calculated based on the percent conversion of the pulse labeled core-glycosylated form (cg-form, filled arrowhead) into the complex-glycosylated form (fg, empty arrow head) of hERG-G601S after 3 h of chase (<i>n</i> = 4). <b>(F)</b> ³⁵S-methinonine and ³⁵S-cysteine incorporation into newly synthesized hERG-G601S during the 20 min pulse labeling at 26°C (<i>n</i> = 4). *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–4 independent experiments. The underlying data of panels A–F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

<i>RPL12</i> knockdown increases the stability of the core- and complex-glycosylated forms of ΔF508-CFTR.

<p><b>(A, B)</b><i>RPL12</i> ablation increases the conformational stability of the solubilised rΔF508-CFTR. The thermoaggregation propensity of rΔF508-CFTR as a surrogate indicator of the channel conformational stability was determined in cell lysates of HeLa cells transfected with <i>RPL12</i> or NT siRNA in comparison to WT-CFTR. To minimize the amount of core-glycosylated form (filled arrowhead), cells were treated with CHX (2 h, 100 μg/ml) prior to lysis. Cell lysates were incubated for 15 min at 20°C–80°C followed by the sedimentation of aggregates and visualizing the remaining soluble channels by immunoblotting (A). The complex-glycosylated channel (empty arrowhead) was quantified by densitometry (B, <i>n</i> = 3–5). <b>(C)</b> Stability of ΔF508-CFTR after (left panel) or without (right panel) low-temperature rescue in HeLa cells upon <i>RPL12</i> knockdown was determined by immunoblot with CHX chase. <b>(D, E)</b> The complex-glycosylated (D, open arrowhead in C) or core-glycosylated CFTR (E, filled arrowhead in C) disappearance was quantified by densitometry and is expressed as percent of the initial amount (<i>n</i> = 3). <b>(F, G)</b> The effect of <i>RPL12</i> silencing on the PM stability (F, <i>n</i> = 4) and functional stability (G, n = 3) of rΔF508-CFTR after 1.5 and 3 h chase at 37°C. Same values as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.g002" target="_blank">Fig 2E and 2F</a> depicted as chase time-dependent percent remaining. <b>(G)</b> The effect of <i>RPL12</i> knockdown on the stability of metabolically labeled core-glycosylated ΔF508-CFTR in CFBE. Labeling was performed for 3 h at 26°C followed by chase for 2 h at 37°C. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–5 independent experiments. The underlying data of panels B and D–H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

Slow-down of translational elongation rate partially corrects ΔF508-CFTR folding defects.

<p><b>(A, B)</b> Schematic depiction of the run-off elongation experiment (A) and relative translation elongation rates in HeLa cells transfected with <i>RPL12</i> or NT siRNA (B). The values were normalized to cells in which the initiation was stopped with harringtonine (2 μg/ml), but the run-off elongation was allowed to proceed without CHX inhibition (<i>n</i> = 4). <b>(C, D)</b> PM density and total expression of ΔF508-CFTR after 24 h treatment with increasing concentrations of CHX (C) or emetine (D) in HeLa. ΔF508-CFTR expression was visualized by immunoblot using anti-HA antibody, anti-β-actin antibody served as loading control (upper panels). Densitometric analysis of core-glycosylated ΔF508-CFTR expression and PM density are expressed as a percentage of DMSO controls (lower panels, <i>n</i> = 3). *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–4 independent experiments. The underlying data of panels B–D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

Genes involved in the function of the cytoplasmic exosome, nuclear exosome, rRNA processing and ribosome influence the function of Yor1-ΔF670.

<p><b>(A)</b> Box-whisker plot of the cell proliferation parameter L (time to reach half-maximal carrying capacity) of the <i>yor1-Δ0</i> (gene deletion) strain (cyan) and the <i>yor1-ΔF670</i> mutant (magenta) following treatment with oligomycin at multiple concentrations. 287 cultures were analyzed for <i>yor1-Δ0</i> and 384 cultures for <i>yor1-ΔF670</i>. “N < 287” or “N < 384” indicates that the concentration of oligomycin was fully growth inhibitory for some of the replicate cultures. Box plots indicate the central 75% of values, whiskers the total value range and averages are indicated by black bars. At higher growth inhibitory concentrations, the phenotypic distributions widen, and thus the high range for the <i>yor1-Δ0</i> at 0.15 ug/mL oligomycin is not depicted to permit better visualization of the data. The single mutant reference strain data from panel A is dose-normalized and plotted in the background for the identically normalized double-mutant data (black dots) in panels B–E to illustrate how each gene deletion influences the oligomycin resistance associated with the <i>yor1-Δ0</i> and <i>yor1-ΔF</i> alleles. <b>(B)</b> The <i>RPL12A</i> deletion increases oligomycin resistance in the context of <i>yor1-ΔF670</i> (magenta), but not <i>yor1-Δ0</i> (cyan). Oligomycin resistance was compared between the single mutants (shown in panel A) and the respective double mutant cultures, separately constructed in quadruplicate (biological replicates, black circles). Data for each series of replicates was normalized by the difference in L in the absence of oligomcyin (orange circles). <b>(C–E)</b> Plots similar to those in panel B are shown for gene modules comprising the cytoplasmic exosome involved in mRNA regulation (C), genes functioning in the nuclear exosome and rRNA processing (D), and additional ribosomal proteins (E). See <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.t001" target="_blank">Table 1</a></b> for additional information. The underlying data of panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

Knockdown of the human homologs of Yor1-ΔF670 modifier genes increases the biochemical and functional expression and stability of the low temperature rescued ΔF508-CFTR.

<p><b>(A)</b> PM density of rΔF508-CFTR (48 h, 26°C) after 1 h chase at 37°C was determined by cell surface ELISA in human CFBE, expressing CFTR under the control of tetracycline inducible transactivator and is expressed as percent of wild-type (WT) CFTR. Indicated genes were silenced with two or three individual siRNAs, and the average PM density is shown as red line. NT siRNA served as negative and the corrector VX-809 as positive controls (<i>n</i> = 3–6). <b>(B)</b> Schematic depiction of the assay (upper panel) and representative traces (lower panel) of rΔF508-CFTR function assayed by halide-sensitive YFP quenching in CFBE cells. Knockdown was achieved with two individual siRNAs per indicated gene, and the measurement was performed after 1.5 h chase at 37°C. The ΔF508-CFTR function was measured by determining the YFP quenching kinetics in response to extracellular iodide addition in the presence of forskolin (10 μM), IBMX (250 μM), cpt-cAMP (250 μM), and genistein (50 μM). <b>(C)</b> The effect of the indicated gene knockdown on the function of rΔF508-CFTR after 1.5 h chase at 37°C as determined by halide-sensitive YFP quenching (<i>n</i> = 3). <b>(D)</b> Correlation between the PM density and functional increase of rΔF508-CFTR after knockdown of Yor1-ΔF670 modifier homologs as determined in panels A and C. <b>(E, F)</b> The effect of knockdown with two individual siRNAs per gene on PM stability (E, <i>n</i> = 4) and functional stability (F, <i>n</i> = 3) of rΔF508-CFTR after 1.5 and 3 h chase at 37°C. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. Error bars indicate standard error of the mean (SEM) of 3–6 independent experiments. The underlying data of panels A, C, E, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

<i>RPL12</i> knockdown increases the conformational maturation of ΔF508-CFTR at the ER.

<p><b>(A)</b> Determination of ΔF508-CFTR translation and ER folding efficiency by metabolic pulse chase in CFBE and HeLa cells transfected with <i>RPL12</i> or NT siRNA. Labeling with [³⁵S]-methionine and [³⁵S]-cysteine was performed to measure translation (30 min in CFBE or 20 min in HeLa at 26°C) or maturation efficiency (for 3 h at 26°C followed by 2 h chase at 37°C) in duplicates. <b>(B)</b> The folding efficiency of ΔF508-CFTR after 2 h chase at 37°C in CFBE (left upper panel) and HeLa cells (right upper panel) was determined by calculating the percent of pulse-labeled immature, core-glycosylated ΔF508-CFTR (B-band, filled arrowhead in A) conversion into the mature, complex-glycosylated form (C-band, empty arrowhead in A) (<i>n</i> = 4–6). The total labeling for 3 h was extrapolated from values obtained for 20 or 30 min pulse labeled samples. Quantitative analysis of ³⁵S-methionine and ³⁵S-cysteine incorporation during the 20 or 30 min labeling period at 26°C into the newly formed ΔF508-CFTR in CFBE (left lower panel) and HeLa cells (right lower panel) (<i>n</i> = 3). <b>(C)</b> [³⁵S]-methionine and [³⁵S]-cysteine incorporation during the labeling period at 37°C into the newly formed ΔF508-CFTR in CFBE (left panel, 30 min) and HeLa cells (right panel, 20 min) (<i>n</i> = 3). <b>(D)</b> Determination of ΔF508-CFTR translation and ER folding efficiency by metabolic pulse chase in HeLa cells transfected with <i>RPL12</i> or NT siRNA with or without VX-809. Labeling was performed for 20 min with no chase (0 h chase) or for 3 h followed by 2 h chase, both at 37°C. The folding efficiency of ΔF508-CFTR after 2 h chase at 37°C is depicted in the lower panel (<i>n</i> = 3) and was calculated based on the extrapolated total labeling for 3 h from values obtained for the 20 min pulse labeled samples without chase. Radioactivity was quantified based on phosphoimage analyses and not by densitometry of the autoradiographs used for illustration. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Error bars show SEM of 3–6 independent experiments. The underlying data of panels B–D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

Yeast deletion suppressors of Yor1-ΔF670 grouped by functional module with human homologs and functional annotations.

<p>Yeast deletion suppressors of Yor1-ΔF670 grouped by functional module with human homologs and functional annotations.</p

SiRNA-mediated silencing of ribosomal stalk proteins enhances the PM density, function, and stability of ΔF508-CFTR.

<p><b>(A, B)</b> The effect of ribosomal stalk protein (RPLP0—P0, RPLP1—P1, and PRPL2—P2) and eEF-2 knockdown on the PM density (A) and function (B) of rΔF508-CFTR in CFBE. The indicated ribosomal proteins were silenced with two to three individual siRNAs and the mean PM density or function is shown as a red line. The values are expressed as percent of NT siRNA controls (<i>n</i> = 3). <b>(C, D)</b> The effect of ribosomal stalk protein and eEF-2 knockdown on the PM (C, <i>n</i> = 3) and functional stability (D, <i>n</i> = 3) of rΔF508-CFTR after 1.5 and 3 h chase at 37°C.*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Error bars show SEM of three independent experiments. The underlying data of panels A–D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p

HGS expression in <i>Hgs</i>^<i>+/+</i> and <i>Hgs</i>^<i>tn/tn</i> tissues.

<p>(A) qPCR analysis of <i>Hgs</i> mRNA expression in 4-week-old <i>Hgs</i>^<i>+/+</i> tissues. Transcript level is expressed relative to <i>Hgs</i> level found in the brain. (B) Representative immunoblot of HGS expression in 4-week-old <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) mice. β-actin was used as a loading control. (C) qPCR analysis of <i>Hgs</i> levels from the brains of <i>Hgs</i>^<i>+/+</i> mice during postnatal development. (D) Representative immunoblot analysis of HGS expression from embryonic day 15 (E15) to postnatal day 35 (P35) in <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) brain lysates. β-tubulin is used as a loading control. (E) Quantitation of developmental time course of HGS expression in <i>Hgs</i>^<i>+/+</i> (wt) and <i>Hgs</i>^<i>tn/tn</i> (<i>tn</i>) mice expressed as percent of E15 <i>Hgs</i>^<i>+/+</i> levels. Symbols represent unpaired t-tests corrected for multiple comparisons using the Holm-Sidak method. A one way anova with a Geisser-Greenhouse adjustment demonstrated a significant difference between time points. (F) Quantitation of HGS expression in <i>Hgs</i>^<i>tn/tn</i> mice expressed as a percent of <i>Hgs</i>^<i>+/+</i> controls at each developmental time point. Data are shown as ± SE. Symbols represent unpaired t-tests. *p<0.05 and ***p<0.001.</p