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

Investigating the consequences of human disease related mutations on 60S ribosome assembly and function in yeast

In actively growing yeast cells, thousands of ribosomes are synthesized per minute. After rapid assembly, these complex machines must faithfully translate mRNA into the cell’s proteome, making it crucial that immature and functionally defective ribosomal subunits do not enter the translating pool. The eukaryotic ribosome biogenesis pathway is a complex series of assembly events that involves the transcription and processing of ribosomal RNA along with its folding and assembly with approximately 80 ribosomal proteins. This pathway includes quality control checkpoints, including a translation-like “test-drive” of the large (60S) subunit during final cytoplasmic maturation. In this work, I use the budding yeast Saccharomyces cerevisiae as a model to study the consequences of mutations in genes associated with 60S quality control. I show that a mutation associated with T-cell acute lymphoblastic leukemia, rpl10-R98S, blocks the final two steps of large subunit ribosome maturation in yeast. Through my analysis of rpl10-R89S, I provide important new insights into mechanisms involved in 60S maturation in the cytoplasm, and demonstrate that the major 60S quality control checkpoint can be bypassed through multiple pathways.Microbiolog

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics

<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

<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

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

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

Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis

Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis.status: publishe