Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

Abstract

Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

Keywords: IRES; RNA; eukaryotic translation; single molecule.

FIGURE 1.

Single-ribosome translation assay: design, ribosome location on mRNA, and fluorescent labeling. (A) The mRNA/ribosome complex is attached to a PEG–neutravidin-coated coverslip through a biotinylated probe hybridized to the 5′ part of the mRNA. mRNA is labeled with the fluorescent probes (UP- and DOWN-primers). (B) Initial locations of the ribosomal A, P, and E sites are indicated. The curved dashed line represents the extremity of the ribosome when bound to the IRES. The IRES initiator codon located at the A-site is indicated in red. To prevent any influence of the ATTO-dye on the probes’ annealing efficiency, three noncomplementary nucleotides have been added to the probes (in blue). Dashed lines represent the position of the ribosome extremity for which 100% of each primer is detached. (***) The first codon read by a tRNA after the translocation of PKI of the IRES from A to P site. (C) Zoomed-in view of single-molecule TIRFM images of UP-(+5)-R primers (left), DOWN-(+14)-G-primers (center). Overlay (right) shows colocalization in yellow. Images are background-filtered using imageJ. (D) Same single-molecule TIRFM images as in C using a mRNA where the DOWN-primer (top line) or UP-primer (bottom line) binding site sequence is replaced by its complementary sequence. Signal scale is the same for the two images of each channel.

FIGURE 2.

Specific binding of the ribosome to the IRES. For each gel shift assay, concentrations of R-(UP)+5-primers, mRNA, and ribosomal subunits (40S and 60S) are indicated. Mobility shifts were visualized using the primer fluorescence, as indicated in Materials and Methods. (A) Increasing the concentration of ribosomal subunits induces a gel shift only in the presence of mRNA. The 30 nM concentration of ribosomal subunits is selected for further experiments. (B) Increasing the concentration of a nonspecific RNA competitor (tRNA) does not change the gel shift. (C) Increasing the concentration of a specific but nonfluorescent RNA competitor (CrPV IRES) reduces the binding of the ribosomal subunits to the mRNA. Images have been obtained by fluorescence scanning with a 633 nm laser and a 670BP30 filter (Typhoon, GE Healthcare). Contrast and brightness have been modified if necessary using Photoshop CS6. These changes have been applied equally across the entire images.

FIGURE 3.

Analysis of the UP- and DOWN-primers’ departure times on CrPV and HCV IRES mRNAs. (A) Fraction of remaining R-UP-(+5) primers versus time for the CrPV IRES-mediated translation experiment (red), controls using cycloheximide (blue), an mRNA without IRES (purple), and photobleaching (gray). t = 0 is the RRL arrival time at the imaged area. Errors bars are the standard deviation of all the curves obtained for each image sequence (seven sequences for translation, six for ΔIRES control, three for cycloheximide control, 12 for photobleaching). (B) Orange curve: histogram showing the fraction of colocalized spots (in %, y-axis) from which G-DOWN-(+14) and R-UP-(+5) primers depart with a given time interval (in sec, x-axis). Black curve: Gaussian fit of this histogram. (C) Fraction of R-UP-(+5) primers disappearing at a given time (red markers) and fit using a two-parameter model (red solid line). Comparison with G-UP-(+5) primers disappearing at a given time (black markers) in a control experiment without 80S preincubation, with the corresponding fitting curve (black solid line). All experiments were performed with preincubated ribosomes, except for the control in panel C. (D) Fraction of remaining R-DOWN-(+24) primers versus time for the HCV IRES-mediated translation experiment (red), controls using HCV-IRES mRNA with a STOP codon before the DOWN binding site (brown), and an mRNA without IRES (purple). t = 0 is the RRL arrival time at the imaged area, time between two images is 2.5 sec. Errors bars are the standard deviation of all the curves obtained for each image sequence (five sequences for translation, nine for mRNA with STOP control, five for ΔIRES control). (E) Fraction of R-UP-(+24) primers disappearing at a given time (red markers) and fit using a two-parameter model (red solid line). In panels B,C, and E, correction was performed using mRNA without IRES and the plotted curves represent the average curves of the bootstrapping procedure described in Materials and Methods, with error bars given by the standard deviation of the bootstrapped samples.

FIGURE 4.

Model for first translocation cycles from the CrPV IRES. Translocation of the PKI of the IRES (green part) from A- to P-sites is a very slow step catalyzed by eEF2. The free A-site is mostly filled by a cognate tRNA^ALa at the alanine codon (***) (when translation initiates in the 0 frame). From our data, we cannot exclude that the next elongation cycle also occurs slowly, due to the presence of the IRES in P- and E-sites (down panel). Elongation then resumes at high speed (1.4 sec). All subsequent steps occur at the same speed, indicating that IRES no longer impacts the functioning of the ribosome.