PMC

RPS25-SNAP restores HCV IRES-mediated translation in RPS25 KO cells. Hap1, RPS25 KO #1, and KO #1 + RPS25-SNAP cells were electroporated with a dual-luciferase construct containing the HCV IRES. Renilla and firefly luciferase were measured and plotted as the Renilla/firefly ratio on the y axis. The values from three independent experiments were averaged, and the error bars represent the SEM.

Kinetics of the 40S subunit recruitment to HCV IRES. (A) Example of a trace. Binding of the 40S subunit was detected by the appearance of FRET. (B) Representative 40S subunit arrival times distribution at 40 nM. Non-single exponential behavior is apparent. (C) Representative lifetime distribution at 40 nM 40S subunit. (D) Arrival rates were linearly dependent on the small subunit concentrations (E), whereas residency times were concentration-independent. k_obs, measured reaction rate. Error bars represent error of the fit. The numbers of molecules used to obtain these fits are 75, 148, 162, and 157, correspondingly.

SNAP-tag on RPS25 does not interfere with ribosome function. Cell lysates from KO #1 + RPS25-SNAP were treated with cycloheximide (Left) or puromycin (Right) and separated by sucrose gradient ultracentrifugation. Sucrose gradients were fractionated with the gradient fractions indicated on the x axis, and absorbance at 254 nm (black line) was continuously measured. Absorbance values are expressed as arbitrary units (AU). Proteins were precipitated from each gradient fraction and separated by 12% (wt/vol) SDS/PAGE. Gels were scanned for fluorescence, and fluorescence intensities were quantified and expressed as a percentage of the total fluorescence from all fractions (red line). RPS25-SNAP, RPS6, and RPL13a were detected by immunoblotting.

Expression of RPS25-SNAP in cell lines depleted of RPS25 using CRISPR-Cas9–mediated deletions in the RPS25 genomic sequence. (A) Genome sequences of the exonic and intronic sequences surrounding the ATG translation start codon (highlighted in red) from WT and CRISPR RPS25 KO cells. (B) Levels of RPS25 protein were undetectable by immunoblotting in two isolated RPS25 KO cell lines. Following lentivirus transduction with RPS25-SNAP, RPS25 was detectable by immunoblotting as a 43-kDa fusion protein. RPS6 and RPL13a were used as loading controls. MW, molecular weight. (C) RPS25 KO and add-back cell lines expressing RPS25-SNAP were lysed in the absence (lanes 1 and 4) or presence (lanes 2 and 5) of fluorescent SNAP649 dye. Cells were incubated in media containing 5 μM SNAP-fluorescein dye before lysis (lanes 3 and 6). Expression of RPS25-SNAP, RPS6, and RPL13a was detected by immunoblotting, and labeled RPS25 was detected by fluorescent scanning of the SDS/PAGE gel.

Conformational dynamics of 40S:HCV IRES complexes. The 40S:IRES complexes were formed in bulk, immobilized on the surface of the microscope slide, and illuminated with a 532-nm laser. (A) Composite image of 40S subunit (green) and HCV IRES (red) fluorescence channels collected at green illumination conditions. An area of approximately 1,000 μm² is shown. (B) Example of a fluorescence trace. The anticorrelated behavior of the green and red signals is a signature of the FRET. The transition from low to high FRET occurs at a mark of ∼40 s. (C) 40S:HCV IRES FRET intensity histogram. Gaussian fits are shown in red, and the sum of individual fits is shown in gray (n = 146 molecules used to build distribution). (D) Control histogram of 40S subunit fluorescence bleed-through (n = 328). (E) Control histogram of 40S:HCV IRES complexes, where HCV IRES was labeled with scrambled Cy5 oligonucleotide (n = 128). (F) 40S:HCV IRES FRET intensity histogram in the presence of diluted translational extract (n = 111).