RACK1 on and off the ribosome

Abstract

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any RP from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular nonessentiality from yeast to humans, RACK1 readily reassociates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight into the biochemical basis of RPs exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

Keywords: IRES; RACK1; human; ribosome; single-molecule fluorescence; translation.

FIGURE 1.

RACK1-ybbR incorporates into translating ribosomes in human cells. (A) Structure of the human ribosome (PDB 6ek0). The 40S (tan) and 60S (blue) ribosomal subunits, RACK1 (pink), mRNA (gray line), and an E-site tRNA (black) are indicated. The WD40 repeat structure of RACK1 is illustrated in the inset with a ribbon depiction (also from PDB 6ek0). There are one and three unresolved amino acids on the amino- and carboxy-termini of RACK1, respectively. (B) Plot of the proliferation of the indicated cell lines as measured by an MTT cell proliferation assay relative to the wild-type cells at day 4. Error bars represent the 95% CI (n = 10). (C) UV absorbance trace (at 254 nm) of polysome profiling and associated western blot analysis of the RACK1-ybbR cell line. “Mono” and “poly” refer to peaks that correspond to monosomes and polysomes, respectively.

FIGURE 2.

Labeling ribosome-bound and recombinant RACK1-ybbR with fluorophores using SFP synthase. (A) Model structure of the human 40S ribosomal subunit (tan) bound to the HCV IRES (gray), with RACK1 highlighted in pink (PDB 5a2q). Key locations on the ribosome and IRES are indicated, with this view focused on the solvent-exposed surface of the 40S subunit. (B) Model of human RACK1 structure with the carboxy-terminal ybbR peptide tag indicated. RACK1-ybbR was site-specifically labeled by incubation with SFP synthase and CoA-Cy3 or CoA-Cy5 conjugated dye substrates. (C) Image of a representative gel that was first scanned for Cy5 fluorescence (right) and subsequently stained with Coomassie blue (left) following SDS-PAGE analysis of RACK1-ybbR post-labeling with and without SFP synthase. (D) Image of a representative gel that was first scanned for Cy5 fluorescence (right) and subsequently silver stained (left) following SDS-PAGE analysis of the indicated 40S ribosomal subunits, with the RACK1 bands indicated by arrows on both images.

FIGURE 3.

RACK1-ybbR is proximal to domain II of the HCV IRES and provides a signal for 40S-IRES association and conformational dynamics on two single-molecule fluorescence platforms. (A) Structural model of the human 40S ribosomal subunit (tan) bound to the HCV IRES (gray), with RACK1 highlighted in green (PDB 5a2q). The carboxyl terminus of RACK1, and the locations of C44 and U56 in the HCV IRES are highlighted, with the measured distance between them indicated. (B) Representative single-molecule fluorescence trace and FRET conversions from TIRFM experiments, with FRET between RACK1-ybbR-Cy3 and IRES(C44-Cy5) highlighted in gray. smFRET was signified by anticorrelated changes in fluorescence intensity of the fluorophores, and single 40S-IRES complexes were identified via a single-step photobleach in the Cy3 donor signal, indicated by the arrow. (C) Schematic of surface immobilized 40S-RACK1-ybbR-Cy3:Cy5-HCV-IRES complexes on a quartz slide used for TIRFM. (D) Cumulative distribution plot of observed FRET intensities for 40S-RACK1-ybbR-Cy3 with IRES(Cy5-U56, purple) or IRES(Cy5-C44, orange) in the presence of 2 mM (solid lines) or 5 mM Mg²⁺ (dashed lines). The number of traces analyzed (n) for panels D are listed in Supplemental Figure 4I. (E) Schematic of 40S-RACK1-ybbR-Cy5 delivery to surface-immobilized HCV IRES labeled at C44 with Cy3 or Cy3.5 in zero-mode waveguides (ZMWs). The blue oval is the full view of an RS chip that has both IRES(Cy3-C44) and IRES(Cy3.5-C44) immobilized in individual ZMWs on the chip. Below the schematic are example single-molecule fluorescence traces that depict Cy3-Cy5 (left) or Cy3.5-Cy5 (right) upon delivery of 40S-RACK1-ybbR-Cy5 to dual-immobilized IRES(Cy3-C44) and IRES(Cy3.5-C44). The respective FRET events are highlighted in gray, and photobleaching of the donors are indicated by the arrows. (F) Schematic of an individual ZMW showing delivery of 40S-RACK1-ybbR-Cy5 to a Cy3.5 labeled HCV IRES. (G) Cumulative distribution plots of observed FRET efficiency from 40S-RACK1-ybbR-Cy5 FRET with either IRES(Cy3-C44) (green) or IRES(Cy3.5-C44) (yellow) after delivery to dual-immobilized IRESs. The number of traces analyzed (n) for panel G are listed in Supplemental Figure 5D.

FIGURE 4.

Monitoring RACK1 flux by real-time smFRET in ZMWs. (A) Schematic of RACK1 delivery to surface-immobilized ΔRACK1 40S in complex with the HCV IRES labeled at C44 with Cy3.5 in ZMWs. (B) Representative single-molecule fluorescence trace for RACK1-ybbR-Cy5 binding to a single ΔRACK1 40S:Cy3.5-IRES complex indicated by Cy3.5-Cy5 FRET, highlighted in gray. (C) Cumulative distribution plot of observed RACK1 association times from 0 to 60 sec. The labels on the right indicate RACK1-ybbR-Cy5 concentration (nM). The lines represent fits to double-exponential functions. (D) Plot of observed RACK1 association rates (k_obs) of the fast phase at the indicated concentrations. Error bars represent the 95% confidence interval for each value. The line represents a fit to a linear function, with equation y = 0.0017x + 0.0149 and R² = 0.9225, which yielded the indicated association rate (k_on) for the fast phase. The nonzero intercept may be due to experimental factors such as the presence of unlabeled RACK1-ybbR that competes with the labeled protein (RACK1-ybbR had ∼70% labeling efficiency). (E) Plot of observed association rates (k_obs) for the fast phase at the indicated temperatures upon delivery of RACK1-ybbR-Cy5 at 75 nM. Error bars represent the 95% confidence interval for each value. (F) Plot of the observed number of binding events on a single ΔRACK1 40S-IRES complex within a single experiment, indicated by smFRET, for RACK1-ybbR-Cy5 and Asc1p-ybbR-Cy5 delivered at 15 or 75 nM at the indicated temperatures. (G) Native gel electrophoresis analysis of 40S-RACK1-ybbR-Cy5 at 40 nM following competition for 24 h with recombinant unlabeled RACK1-ybbR and Asc1p-ybbR at 20-fold excess, and recombinant RACK1-ybbR-Cy3 at 10-fold excess. The gels were analyzed in parallel and are displayed as an overlay of Cy5 (red) and Cy3 (green) fluorescence. The integrated density of each band was quantified using ImageJ, normalized to the respective control sample (lane 1, from left), and the mean (n = 3) Cy5 fluorescence was plotted. Error bars represent standard deviation, and circles represent individual data points. (H) Native gel electrophoresis analysis of 40S-RACK1-ybbR-Cy5 at 40 nM following competition after the indicated times with recombinant unlabeled RACK1-ybbR at 20-fold excess. A representative gel is shown and displays Cy5 fluorescence. Each point on the graph below represents the mean fluorescence intensity remaining relative to the 0.08 h time point, and error bars represent standard deviation. The line represents a fit to an exponential function (R² = 0.93), which yielded t_1/2 = 15 h ± 5 h. The number of traces analyzed (n) for panels C, E, and F are listed in Supplemental Figures 9E, 10C.