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.Ribosomopathies are a family of congenital diseases that are linked to genetic defects in ribosomal proteins or ribosome biogenesis factors. They are characterized by pleiotropic abnormalities that include birth defects, heart and lung diseases, connective tissue disorders, anemia, ataxia, and mental retardation (reviewed in ref. 1). Although each ribosomopathy presents a unique pathological spectrum, the inherited forms are characterized by bone marrow failure and anemia early in life, followed by elevated cancer risk by middle age. For example, although childhood anemia is one of the cardinal symptoms of the genetically inherited disease Diamond–Blackfan anemia, these patients have a fivefold higher lifetime risk of cancer than the general population and a 30- to 40-fold higher risk of developing acute myeloid leukemia, osteosarcoma, or colon cancer (reviewed in refs. 2, 3). Similarly, patients with X-linked dyskeratosis are predisposed to myeloid leukemia and a variety of solid tumors (4), whereas patients with 5q− syndrome are at higher risk of developing acute myeloid leukemia (reviewed in ref. 5). In the genetically tractable zebrafish model, heterozygous loss-of-function mutations in several ribosomal proteins cause development of peripheral nerve sheet tumors (6). Somatically acquired mutations in ribosomal proteins are also implicated in cancer: ∼10% of children with T-cell acute lymphoblastic leukemia (T-ALL) were found to harbor somatic mutations in the ribosomal protein of the large subunit (LSU) 10, 5, and 22 (RPL10, RPL5, and RPL22) (7). [Note that the proteins encoded by these genes are also named uL16, uL18, and eL22, respectively, under the newly proposed uniform ribosomal protein nomenclature (8).] A separate study identified heterozygous deletions in the region of chromosome 1p that contains RPL22 (eL22) in an additional 10% of patients with T-ALL (9). The model of ribosomal proteins as targets for somatic mutations in cancer is further supported by the finding that two ribosomal protein genes (RPL5/uL18 and RPL22/eL22) are included in the list of 127 genes identified as significantly mutated in cancer in the context of the first Cancer Genome Atlas pan-cancer analysis in 12 tumor types (10).Ribosomopathies present an intriguing paradox: Although patients initially present with hypoproliferative disorders (e.g., anemias, bone marrow failure), those who survive to middle age often develop hyperproliferative diseases (i.e., cancers). The link between ribosome defects and hypoproliferative disease phenotypes has been extensively studied: The current working hypothesis is that impaired ribosome biogenesis activates a “ribosomal stress” cascade, activating the cellular TP53 pathway and resulting in cell cycle arrest and cell death (11). However, activation of TP53 does not explain why ribosomal defects are associated with hyperproliferative diseases, particularly cancer. Mutations in the ribosomal protein gene RPL10/uL16 were recently identified in patients with T-ALL (7). The T-ALL–associated RPL10/uL16 mutations occurred almost exclusively in residue arginine 98 (R98), with the exception of one patient harboring the Q123P mutation, which lies adjacent to R98 within the rpL10/uL16 3D structure (Fig. 1). Both residues are at the base of an essential flexible loop in rpL10 that closely approaches the peptidyltransferase center in the catalytic core in the ribosome (12). In addition to its role in catalysis (13, 14), rpL10/uL16 plays an important role in the late stages of 60S subunit biogenesis. After initial production of the separate ribosomal subunits in the nucleus, immature and functionally inactive pre-60S subunits are exported to the cytoplasm, where they undergo additional maturation events (15), including incorporation of rpL10/uL16, before they can associate with mature 40S subunits and engage in protein synthesis (16). Among the critical set of final 60S maturation steps is the release of the antiassociation factor Tif6, followed by release of Nmd3, the primary export adaptor for the pre-60S subunit in yeast and in humans (17, 18). Tif6 release requires the tRNA structural mimic Sdo1p (19) and the GTPase Efl1, a paralog of eukaryotic elongation factor 2 (eEF2) (20). We have suggested that structural rearrangements of the internal loop of rpL10/uL16 coordinate this final maturation process, resulting in a test drive of the pre-60S subunit to ensure that only properly functioning subunits are allowed to enter the pool of translationally active ribosomes (13, 21). Defective ribosomes carrying mutations in rpL10/uL16 specifically fail in this test drive, leading to their degradation through a molecular pathway that is yet to be characterized. Beyond 60S maturation, rpL10/uL16 plays an important role in coordinating intersubunit rotation and controlling allosteric rearrangements within the ribosome, helping to ensure the directionality and fidelity of protein synthesis (13).Open in a separate windowFig. 1.Localization of rpL10 and the loop in the LSU. (A) rpL10/uL16 in the context of the crown view of the LSU. (B) Close-up view of rpL10/uL16 and the local environment. The flexible loop structure is indicated by dashed red lines, and the positions of R98 and Q123 are indicated. rpL10/uL16 is situated between helices 38 and 89, and it is located in close proximity to several functional centers of the LSU, including the peptidyltransferase center (PTC), aa-tRNA accommodation corridor, and elongation factor binding site. Images were generated using PyMOL.rpL10/uL16 is highly conserved among eukaryotes: The yeast and human proteins are interchangeable, and residue 98 is invariantly an arginine (22). Human RPL10/uL16 is located on the X chromosome, and is therefore expressed as a single-copy gene in males. Thus, the haploid yeast model is an excellent mimic of the situation in the cells of a patient with T-ALL. Yeast cells expressing rpl10-R98S, rpl10-R98C, and rpl10-H123P (corresponding to Q123 in human rpL10/uL16) as the sole forms of rpL10/uL16 displayed proliferative defects. Further, polysome profiling revealed increased ratios of free 60S and 40S subunits vs. monosomes, markedly reduced polysomes, and the presence of halfmers in these mutants, suggesting defects in both ribosome biogenesis and subunit joining (7). Tif6 and Nmd3 both accumulated in the cytoplasm in the mutant cells, indicating a defect in their release from the cytoplasmic 60S (7). Thus, all of the rpl10/uL16 mutations appeared to affect 60S biogenesis at the Efl1-dependent quality control step. Consistent with the yeast-based observations, mouse lymphoid cells expressing rpl10-R98S displayed slower proliferation rates than cells expressing WT RPL10/uL16 and conferred defective polysome profiles (7).The studies presented in the current report use the yeast rpl10-R98S mutant to elucidate the structural, biochemical, and trans-lational fidelity defects that may lead to carcinogenesis. This mutant perturbs the structural equilibrium of ribosomes toward the “rotated state.” At the biochemical level, this underlying structural defect alters the affinity of mutant ribosomes for a specific set of trans-acting ligands. In turn, the biochemical defects affect translational fidelity, promoting elevated rates of −1 programmed ribosomal frameshifting (−1 PRF) and impaired recognition of termination codons. Globally increased rates of −1 PRF result in a decreased abundance of cellular mRNAs that harbor operational −1 PRF signals (23, 24). These −1 PRF signal-containing mRNAs include EST1, EST2, STN1, and CDC13, which play central roles in yeast telomere maintenance (23). In rpl10-R98S cells, the steady-state abundances of these mRNAs are decreased, resulting in telomere shortening. A spontaneously acquired trans-acting mutant suppresses the ribosome biogenesis defects of the rpl10-R98S mutant, thereby reestablishing high levels of ribosome production and cell proliferation. Importantly, however, suppression of the biogenesis and growth impairment defects fails to suppress the profound structural, biochemical, and translational fidelity defects of rpL10-R98S ribosomes. These findings suggest that suppression of the growth defect results from bypassing the test drive. Although the suppressor mutation enables cells to grow at normal rates, genetic suppression comes at the cost of releasing functionally defective ribosomes into the translationally active pool. We propose two different but not mutually exclusive models for how somatically acquired rpL10/uL16 mutations may promote cancer: (i) Mutant ribosomes may drive altered gene expression programs, promoting T-ALL, or (ii) the suppressor mutations may themselves be the drivers of T-ALL.