Mean latencies and their standard deviations (ms) of each S1-evoked potential for AWS and AFS and the results of <i>t</i>-tests.

<p>Mean latencies and their standard deviations (ms) of each S1-evoked potential for AWS and AFS and the results of <i>t</i>-tests.</p

tRNA^Thr(CGU) level is very low in CFBE41o^-, primary human bronchial epithelial (HBE) cells, and pulmonary tissues.

<p>(<b>A</b>) Frequency of the Thr codons per thousand (/1,000) and as absolute numbers (#). (<b>B</b>) Ribosome dwelling occupancy at the ACG codon is highest in CFBE41o^- cells. The graph shows the relationship between dwelling occupancy (normalized codon frequency), as determined by ribosome profiling, and genome codon usage. Two Thr codons, ACG and ACT, are highlighted in black. The dashed line denotes the upper boundary (90% confidence interval) for ribosome dwelling occupancy. (<b>C</b>) Microarray analysis of the absolute tRNA concentration in HeLa cells. Data are means ± SEM (<i>n</i> = 3). tRNA probes are depicted with their cognate codon and the corresponding amino acid; Meti, initiator tRNA^Met. Arrows denote the 2 tRNAs decoding ACG and ACT codons. (<b>D</b>) Comparative microarrays of the abundance of tRNAs^Thr in CFBE41o^- and 4 cystic fibrosis (CF) patient–derived HBE cells relative to HeLa tRNAs for which the absolute concentration of tRNA^Thr is determined in C. Data are means ± SEM (<i>n</i> = 4–5). For the full comparison of the complete tRNA set, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s008" target="_blank">S8 Fig</a>. (<b>E</b>) Northern blot of total tRNA isolated from different cells and human tissues (pulmonary, heart, brain, and kidney) probed with fluorescently labeled tRNA^Thr(CGU) and 5S rRNA probes. HBE denotes primary HBE cells from ΔF508 homozygous CF patients and Pulmonary indicates pulmonary tissue from a non-CF individual. (<b>F</b>) Steady-state CFTR expression in kidney (HEK 293) and neuronal (SH-SY5Y) cell lines. Total expression (i.e., the sum of bands B and C) was normalized to that of NPT and ACTB and was related to wild-type CFTR, which was set to 100%. Data are means ± SEM (<i>n</i> = 3). (<b>G</b>) Comparative microarrays of the abundance of tRNAs^Thr in various tissues and cell lines compared to HeLa tRNAs, which are set as 1 using the same format as in D. The underlying data of panels B–D and F–G can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s014" target="_blank">S1 Data</a>.</p

Synonymous mutations alter cystic fibrosis transmembrane conductance regulator (CFTR) expression.

<p>(<b>A</b>) Quantitative real-time PCR (qRT-PCR) quantification of steady-state CFTR mRNA expression in HeLa and CFBE41o^- cells analyzed 24 h after transfection and normalized to neomycin phosphotransferase (NPT). The mRNA level of wild-type CFTR was set to 100%. (<b>B</b>) Total steady-state CFTR expression (i.e., the sum of bands B and C in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s001" target="_blank">S1B Fig</a>) normalized to that of NPT and β-actin (ACTB) and related to wild-type CFTR, which was set to 100%. In A and B, data are means ± SEM (A: HeLa, <i>n</i> = 3–7; CFBE41o^-, <i>n</i> = 4–9; B: HeLa, <i>n</i> = 5–14; CFBE41o^-, <i>n</i> = 4–7); * <i>P</i> < 0.05, ** <i>P <</i> 0.01, *** <i>P</i> < 0.001 versus wild-type CFTR. (<b>C</b>) Representative immunostaining images (<i>n</i> = 3) of well-differentiated human cystic fibrosis (CF) airway epithelia. The frequency of intracellular versus membrane-localized T2562G-CFTR staining was as follows: 44% of T2562G-CFTR–expressing cells exhibited intracellular staining (12 out of 27) and in 55% (15 out of 27), staining was membrane localized. The perinuclear staining demonstrates the intracellular localization of T2562G-CFTR. The control was non–CFTR-expressing epithelia. β-catenin immunostaining denotes adherens junctions and DAPI—nuclei; scale bars, 10 μm. The underlying data of panels A and B can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s014" target="_blank">S1 Data</a>.</p

T2562G-CFTR exhibits 2 types of channel openings.

<p>(<b>A</b>) Representative single-channel recordings of CFTR variants in excised inside-out membrane patches from CHO cells. For wild-type and T2562C-CFTR, the membrane patches contained 1 channel; for T2562G-CFTR, it contained 2 channels—one with small conductance (sc) and the other with wild-type–like (wtl) openings. Dashed lines indicate the closed channel state and downward deflections correspond to channel openings. For T2562G-CFTR, the indicated 2-s portion is shown on an expanded time scale after filtering digitally. (<b>B–E</b>) Single-channel current amplitude (<i>i</i>), open probability (<i>P</i>_o), mean burst duration (MBD), and interburst interval (IBI) of CFTR variants. Data are means ± SEM (<i>n</i> = 5–9); * <i>P</i> < 0.05 versus wild-type CFTR; ND, not determined. The underlying data of panels B–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s014" target="_blank">S1 Data</a>.</p

Increased tRNA^Thr(CGU) levels ameliorate the expression and stability defects and abolish small-conductance openings of T2562G-CFTR.

<p>(<b>A</b>) Representative northern blot (NB) and WB analysis of HeLa cells cotransfected with tRNA^Thr(CGU) and either wild-type or T2562G-CFTR. (<b>B</b>) Quantification of T2562G-CFTR from A (details in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.g001" target="_blank">Fig 1B</a>) and presented relative to those of wild-type CFTR (set to 100%). Values are means ± SEM (<i>n</i> = 5–7); * <i>P</i> < 0.05; ** <i>P</i> < 0.01. (<b>C</b>) Thermal aggregation propensity assay upon cotransfection with 150 ng tRNA^Thr(CGU) (+tRNA) compared to untransfected controls (-tRNA). Values are means ± SEM (<i>n</i> = 3–5); * <i>P</i> < 0.05 versus wild-type CFTR. (<b>D</b>) Representative single-channel recordings of wild-type, T2562G-, and T2562G-CFTR cotransfected with tRNA (T2562G + tRNA^Thr(CGU)) in excised inside-out membrane patches from CHO cells. The dashed lines indicate where channels are closed and downward deflections of the traces correspond to channel openings. For T2562G-CFTR, the indicated 2-s portion is shown on an expanded time scale after filtering at 50 Hz. For wild-type and T2562G-CFTR coexpressing tRNA^Thr(CGU), membrane patches contained 1 active channel; for T2562G-CFTR, it contained 2 channels—one with sc openings and the other with wtl. (<b>E–H</b>) Single-channel conductance (γ), open probability (<i>P</i>_o), mean burst duration (MBD), and interburst interval (IBI) of the indicated CFTR variants. Data are means ± SEM (<i>n</i> = 6–10); * <i>P</i> < 0.05 versus wild-type CFTR. In E–H, control data are the same as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.g003" target="_blank">Fig 3C–3E</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s005" target="_blank">S5C Fig</a>. The underlying data of panels B–C and E–H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000779#pbio.2000779.s014" target="_blank">S1 Data</a>.</p

The synonymous single nucleotide polymorphism (sSNP) T2562G inverts local translation speed in CFTR mRNA, which can be rescued by tRNA^Thr(CGU).

<p>(<b>A</b>) Thr-854–encoding codon ACT in wild-type CFTR is translated fast, as its cognate tRNA^Thr(AGU) is relatively abundant. (<b>B</b>) T2562G sSNP converts the ACT triplet to ACG codon, which is read by the rare cognate tRNA^Thr(CGU) and reduces local ribosomal speed. Stochasticity in the delivery of tRNA^Thr(CGU) cognate to the ACG codon creates variations in the intimate translation speed of each ACG codon at this position and hence generates 2 distinct CFTR channel populations, one with wild-type–like (wtl) CFTR properties and a second with a reduced conductance and a more compact structure (small-conductance [sc] population). (<b>C</b>) Increase of the cellular level of tRNA^Thr(CGU) pairing to the mutated ACG codon restores ribosome speed at the rare Thr-ACG codon and rescues the CFTR conductance defect.</p