RIBOSOME IN THE BALANCE: STRUCTURAL EQUILIBRIUM ENSURES TRANSLATIONAL FIDELITY AND PROPER GENE EXPRESSION

At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In translation elongation, the binding of two translation elongation factors to the same general region of the ribosome stabilizes them in one of the two extremes of intersubunit rotation; rotated or unrotated. These stabilized states are resolved by expenditure energy in the form of GTP hydrolysis. Here, mutants of the early assembling integral ribosomal protein uL2 (universal L2) are used to test the generality of this hypothesis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium towards the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Shift in structural equilibrium affects the biochemical properties of ribosomes: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented here describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression

The T-cell leukemia related rpl10-R98S mutant traps the 60S export adapter Nmd3 in the ribosomal P site in yeast

Mutations in the ribosomal protein Rpl10 (uL16) can be drivers of T-cell acute lymphoblastic leukemia (T-ALL). We previously showed that these T-ALL mutations disrupt late cytoplasmic maturation of the 60S ribosomal subunit, blocking the release of the trans-acting factors Nmd3 and Tif6 in S. cerevisiae. Consequently, these mutant ribosomes do not efficiently pass the cytoplasmic quality control checkpoint and are blocked from engaging in translation. Here, we characterize suppressing mutations of the T-ALL-related rpl10-R98S mutant that bypass this block and show that the molecular defect of rpl10-R98S is a failure to release Nmd3 from the P site. Suppressing mutations were identified in Nmd3 and Tif6 that disrupted interactions between Nmd3 and the ribosome, or between Nmd3 and Tif6. Using an in vitro system with purified components, we found that Nmd3 inhibited Sdo1-stimulated Efl1 activity on mutant rpl10-R98S but not wild-type 60S subunits. Importantly, this inhibition was overcome in vitro by mutations in Nmd3 that suppressed rpl10-R98S in vivo. These results strongly support a model that Nmd3 must be dislodged from the P site to allow Sdo1 activation of Efl1, and define a failure in the removal of Nmd3 as the molecular defect of the T-ALL-associated rpl10-R98S mutation

<i>TIF6</i> alleles identified as suppressors of <i>rpl10-R98S</i>.

<p><i>TIF6</i> alleles identified as suppressors of <i>rpl10-R98S</i>.</p

<i>NMD3</i> alleles identified as spontaneous suppressors of <i>rpl10-R98S</i>.

<p><i>NMD3</i> alleles identified as spontaneous suppressors of <i>rpl10-R98S</i>.</p

Model.

<p>In the fully closed L1 stalk position, the eIF5A domain of Nmd3 is engaged with the E site, while the eL22-like domain occupies the P site. This position is stabilized by the interaction between the N-terminus of Nmd3 with Tif6. We propose that, following Rpl10 loading, the linkage between Nmd3 and Tif6 is broken, destabilizing the N-terminus of Nmd3 and allowing Nmd3 retraction from the P site and Sdo1 binding.</p

Allele specificity between <i>RPL10</i> and <i>TIF6</i>.

<p><b>A)</b> Structure showing Tif6 bound to Rpl23 on the 60S subunit. Tif6 residue V192 (green) is located at the interface between Tif6 and Rpl23, while Tif6 residues mutated in <i>rpl10-R98S</i> suppressors (magenta) are clustered in a nearby region distinct from the interface with the 60S subunit. (Assembled from PBD file 5ANB) <b>B)</b> 10-fold serial dilutions of AJY3373 (P_GAL<i>-RPL10</i>) containing <i>WT RPL10</i>, <i>rpl10-R98S</i>, or <i>rpl10-S104D</i> vectors, and either empty vector or the indicated alleles of <i>NMD3</i> on centromeric vectors or <i>TIF6</i> alleles on high copy vectors. Cells were spotted onto glucose-containing selective media to repress genomic <i>RPL10</i>. <b>C)</b> Serial dilutions of the glucose repressible <i>EFL1</i> strain AJY2981 (P_GAL<i>-EFL1</i>) containing empty vector or the indicated <i>TIF6</i> or <i>NMD3</i> plasmids. Cells were spotted onto glucose-containing selective media to repress Efl1.</p

The <i>rpl10-R98S</i> defect is not suppressed by Tif6 release.

<p><b>A)</b> The position of Rpl10 in the crown view of the 60S subunit. The central protuberance (CP), helix 38 (H38) and helix 89 (H89) are indicated. A cartoon of Rpl10 structure showing amino acids mutated in T-ALL (blue) (From PDB files 5ANB, 3U5D and 3U5E). <b>B)</b> 10-fold serial dilutions of the glucose repressible <i>RPL10</i> strain AJY3373 (P_GAL<i>-RPL10</i>) harboring <i>WT RPL10</i>, <i>rpl10-S104D</i>, or <i>rpl10-R98S</i> vector, and either empty vector or the indicated <i>TIF6</i> or <i>EFL1</i> plasmids. Cells were spotted onto glucose-containing selective media to repress genomic <i>RPL10</i>. <b>C)</b> Tif6-GFP and Tif6-V192F-GFP localization monitored by fluorescence microscopy in <i>WT RPL10</i>, <i>rpl10-S104D</i>, and <i>rpl10-R98S</i> cells. AJY2766 (P_GAL<i>-RPL10</i>, <i>TIF6-GFP</i>) and AJY3941 (P_GAL<i>-RPL10</i>, <i>TIF6-V192F-GFP</i>) expressing WT <i>RPL10</i>, <i>rpl10-S104D</i>, or <i>rpl10-R98S</i> from plasmids were grown in the presence of glucose to repress genomic <i>RPL10</i>. DIC, differential interference contrast. Scale bar, 5μm.</p

Efl1 activation is inhibited by Nmd3 on <i>rpl10-R98S</i> subunits.

<p><b>A-B)</b> Sdo1 stimulated, 60S-dependent Efl1 GTPase activity was monitored by the release of free phosphate in reactions containing the indicated combinations of 50nM 60S subunits (wild-type or <i>rpl10-R98S</i>), 50nM Efl1, 250nM Sdo1, and the indicated concentrations of Nmd3 in (A) or 250nM Nmd3 in (B). All experiments were done in triplicate, the mean and SD are given. <b>C-D)</b> Nmd3 stimulated, 60S-dependent Lsg1 GTPase activity was monitored by the release of free phosphate in reactions containing the indicated combinations of 50nM 60S subunits (wild-type or <i>rpl10-R98S</i>), 250nM Lsg1, 250nM wild-type Nmd3 or Nmd3-Y379D in (C) or the indicated concentrations of wild-type Nmd3 or Nmd3-Y379D in (D). All experiments were done in triplicate, the mean and SD are given.</p