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. 2006 Feb;13(2):103-11.
doi: 10.1038/nsmb1052. Epub 2006 Jan 22.

Direct ribosomal binding by a cellular inhibitor of translation

Daniel A Colón-Ramos  1 , Christina L Shenvi, Douglas H Weitzel, Eugene C Gan, Robert Matts, Jamie Cate, Sally Kornbluth
Affiliations

Direct ribosomal binding by a cellular inhibitor of translation

Daniel A Colón-Ramos et al. Nat Struct Mol Biol. 2006 Feb.

Abstract

During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES-driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate.

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Figures

Figure 1
Figure 1
Ribosomes restore Reaper’s inhibitory activity. (a) Translation of endogenous RRL mRNAs in the presence or absence of Reaper (Rpr). Biotinylated Reaper peptide or GST protein coupled to streptavidin beads was used to affinity-deplete untreated RRL (containing endogenous RRL mRNA). Translation was assayed and quantitated as described in Methods. (b) Schematic representation of the fractionation protocol used to purify Reaper’s translational inhibitory activity (protocol adapted from refs. 28,29). (c) Translation of endogenous mRNAs as in a, except that RRL was supplemented before the translation reaction with either buffer, fresh RRL, supernatant or polysome pellet (containing polyribosomes and associated initiation factors) (left chart), or buffer, fresh RRL, ribosomal salt wash (RSW) containing initiation factors (IFs) or a ribosomal pellet (right chart). Activity is normalized to that of the affinity-depleted RRL rescued with fresh RRL.
Figure 2
Figure 2
Reaper binds ribosomes through direct interaction with the 40S subunit. (a) Reaper binding to ribosomes. Reaper (or GST)-bound streptavidin beads were used to affinity-deplete RRL (left gel) or Drosophila embryonic extracts (right gel). Bound material was stringently washed, phenol-chloroform extracted and resolved by agarose gel electrophoresis. (b) Reaper binding to tightly associated ribosomal protein S6. RRL was prepared as in a, but bound material was resolved by PAGE and immunoblotted with antibody directed against S6. Input (isolated ribosomes) was also immunoblotted as a control. (c) Reaper binding to 80S complexes. 40S and 60S ribosomal subunits were purified and combined to form 80S complexes, then incubated with biotinylated Reaper, and Reaper-bound material was purified and resolved on denaturing agarose gels (see Methods for details). Addition of Reaper did not cause dissociation of ribosomal subunits. (d) Reaper binding to 80S complexes was assayed as in c, but subunits were either incubated with biotinylated Reaper before 80S reconstitution or stringently washed with high-salt buffer (500 mM KCl) after 80S reconstitution and binding. Stringent high-salt washing conditions resulted in dissociation of 40S and 60S ribosomal subunits, but these same stringent conditions did not affect the 40S-Reaper interaction.
Figure 3
Figure 3
Reaper inhibits translation initiation and induces accumulation of 48S half-mers. (a) Sedimentation profile of normally translating RRL resolved by sucrose-gradient centrifugation. (b) Sedimentation as in a, except that RRL was supplemented with Reaper peptide (final concentration of 40 µM). Note the accumulation of 48S half-mers (arrows) and 80S monosomes.
Figure 4
Figure 4
Reaper does not affect eIF2α phosphorylation or 43S complex formation. (a) Purified 40S subunits and purified eIF3 complex were allowed to interact in the presence or absence of Reaper and resolved on nondenaturing agarose gels. 18S rRNA was visualized by ethidium bromide staining. In the last two lanes (order of addition), either Reaper or eIF3 complex was preincubated for 10 min with the 40S subunit as indicated by the number 1, and the remaining component was then added (number 2). (b) Phosphorylation of eIF2α in the presence or absence of Reaper. Translating RRL not supplemented with hemin, or supplemented with hemin and either a control peptide (Reaper2–16, final concentration of 325 µM) or active Reaper peptide (Reaper16–65 or full-length Reaper, final concentration of 40 µM) was resolved by isoelectric focusing and immunoblotted with antibodies directed against eIF2α to detect eIF2α phosphorylation. Not supplementing RRL with hemin results in activation of HRI kinase, which phosphorylates eIF2α and inhibits translation. (c) Association of eIF2α with 40S subunits in the presence or absence of Reaper. Translating RRL with or without Reaper (final concentration of 40 µM) was resolved by sucrose-gradient centrifugation and fractions were collected. Fractions containing the 40S complex were methanol precipitated, resolved by SDS-PAGE and assayed by immunoblotting using antibody directed against eIF2α. (d,e) Association of eIF4G and eIF4E with the cap structure in the presence or absence of Reaper. Translating RRL extracts with or without Reaper (final concentration of 40 µM) were supplemented and incubated with cap resin (7-methylGpppG resin). Bead-bound material was washed with buffer, resolved by SDS-PAGE and immunoblotted with antibodies directed against eIF4E (d) or eIF4G (e). Free 7-methylGpppG could compete with bound initiation factors from the cap resin (not shown).
Figure 5
Figure 5
Reaper-induced 48S half-mers inefficiently recognize the AUG start site. (a) Toeprinting analyses on the GLA mRNA supplemented with buffer, indicated concentrations of Reaper (Rpr) or pharmacological translation inhibitors. Arrows at left, positions of initiation AUG codons; asterisks at right, positions of toeprinting bands. A dideoxynucleotide sequence generated with the same primer was run in parallel (first four rows). The ribosomal pause sites in the Reaper lanes are not appreciably stronger than those present in the buffer control, but are appreciably weaker than those observed for any of the positive controls. (b) Toeprinting as in a, except that Reaper was added at 15 µM (final concentration) in the presence or absence of cycloheximide.
Figure 6
Figure 6
Reaper induces differential pausing in bicistronic mRNA messages. Shown are toeprinting analyses on the BUN-S bicistronic mRNA supplemented with buffer, varying concentrations of Reaper (Rpr) or cycloheximide. A dideoxynucleotide sequence generated with the same primer was run in parallel (last four rows). Note a titratable increase in ribosomal pausing at the second start site upon addition of increasing amounts of Reaper.
Figure 7
Figure 7
Reaper induces differential inhibition of translation in an mRNA-dependent manner. RRLs were supplemented with saturating levels of either GLA mRNA, HG mRNA or CrPV-EGFP mRNA. In vitro ranslation in the absence or presence of Reaper at the indicated concentrations was performed and quantitated as described in Methods. All reactions were carried out in triplicate. The degree of inhibition by Reaper at a given concentration is dependent on the identity of the mRNA, with CrPV-IRES being expressed at Reaper concentrations that completely inhibit expression of cap-dependent GLA and HG mRNAs.
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Comment in

  • Translation, interrupted.
    Pestova TV, Hellen CU. Pestova TV, et al. Nat Struct Mol Biol. 2006 Feb;13(2):98-9. doi: 10.1038/nsmb0206-98. Nat Struct Mol Biol. 2006. PMID: 16462810 No abstract available.

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