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George Thomas - One of the best experts on this subject based on the ideXlab platform.
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Oncogenic MYC Induces the Impaired Ribosome Biogenesis Checkpoint and Stabilizes p53 Independent of Increased Ribosome Content.
Cancer research, 2019Co-Authors: Carmen Morcelle, George Thomas, Sara C. Kozma, Sandra Menoyo, Francisco D. Morón-duran, Albert Tauler, Antonio GentilellaAbstract:The role of MYC in regulating p53 stability as a function of increased ribosome biogenesis is controversial. On the one hand, it was suggested that MYC drives the overexpression of ribosomal proteins (RP)L5 and RPL11, which bind and inhibit HDM2, stabilizing p53. On the other, it has been proposed that increased ribosome biogenesis leads the consumption of RPL5/RPL11 into nascent ribosomes, reducing p53 levels and enhancing tumorigenesis. Here, we show that the components that make up the recently described impaired ribosome biogenesis checkpoint (IRBC) complex, RPL5, RPL11, and 5S rRNA, are reduced following MYC silencing. This leads to a rapid reduction in p53 protein half-life in an HDM2-dependent manner. In contrast, MYC induction leads to increased ribosome biogenesis and p53 protein stabilization. Unexpectedly, there is no change in free RPL5/RPL11 levels, but there is a striking increase in IRBC complex bound to HDM2. Our data support a cell-intrinsic tumor-suppressor response to MYC expression, which is presently being exploited to treat cancer. Significance: Oncogenic MYC induces the impaired ribosome biogenesis checkpoint, which could be potentially targeted for cancer treatment.
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Abstract 2024: c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC)
Molecular and Cellular Biology, 2015Co-Authors: Suresh Peddigari, Carol A. Mercer, Sara C. Kozma, George ThomasAbstract:We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p53-/- and p53 +/+ Eμ-Myc cells, likely due to the inhibition of global translation. These findings demonstrate a differential abrogation of ribosome biogenesis by partial depletion of two essential RPs, which assemble at a very similar stage in 60S ribosome. For RPL7a, it led to L5/L11/5S-MIC induced p53 mediated cell death, and this was not the case for RPL11 as it is an essential component of the complex. Citation Format: Suresh Peddigari, Carol Mercer, Sara Kozma, George Thomas. c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2015-2024
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abstract 2024 c myc driven b cell lymphomas role of the rpl5 RPL11 5s rrna mdm2 inhibitory complex l5 l11 5s mic
Cancer Research, 2015Co-Authors: Suresh Peddigari, Carol A. Mercer, Sara C. Kozma, George ThomasAbstract:We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p53-/- and p53 +/+ Eμ-Myc cells, likely due to the inhibition of global translation. These findings demonstrate a differential abrogation of ribosome biogenesis by partial depletion of two essential RPs, which assemble at a very similar stage in 60S ribosome. For RPL7a, it led to L5/L11/5S-MIC induced p53 mediated cell death, and this was not the case for RPL11 as it is an essential component of the complex. Citation Format: Suresh Peddigari, Carol Mercer, Sara Kozma, George Thomas. c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2015-2024
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loss of tumor suppressor rpl5 RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity
Molecular and Cellular Biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
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Loss of tumor suppressor RPL5/RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity.
Molecular and cellular biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
Hanna T Gazda - One of the best experts on this subject based on the ideXlab platform.
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Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: Evidence from RPL11-deficient in vivo and in vitro models.
Biochemical and biophysical research communications, 2017Co-Authors: Anirban Chakraborty, Marie-françoise O'donohue, Hanna T Gazda, Tamayo Uechi, Yukari Nakajima, Pierre-emmanuel Gleizes, Naoya KenmochiAbstract:Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of RPL11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, RPL11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of RPL11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency.
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Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes.
Experimental hematology, 2014Co-Authors: Tamara C. Pereboom, Hanna T Gazda, Albert Bondt, Paschalina Pallaki, Timothy D. Klasson, Yvonne J. Goos, Paul B. Essers, Marian J. A. Groot Koerkamp, Frank C. P. Holstege, Lydie Da CostaAbstract:Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.
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frameshift mutation in p53 regulator rpl26 is associated with multiple physical abnormalities and a specific pre ribosomal rna processing defect in diamond blackfan anemia
Human Mutation, 2012Co-Authors: Mee Rie Sheen, Adrianna Vlachos, Mariefrancoise Odonohue, Hanna T Gazda, Milena Preti, Stella M Davies, Antonis KattamisAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ~30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth ribosomal protein regulating p53 activity that is linked to DBA.
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frameshift mutation in p53 regulator rpl26 is associated with multiple physical abnormalities and a specific pre ribosomal rna processing defect in diamond blackfan anemia
Human Mutation, 2012Co-Authors: Adrianna Vlachos, Mariefrancoise Odonohue, Hanna T Gazda, Milena Preti, Stella M Davies, Mee Rie Shee, Antonis KattamisAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA.
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Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-rRNA processing defect in Diamond-Blackfan anemia.
Human Mutation, 2012Co-Authors: Hanna T Gazda, Leana Doherty, Mee Rie Sheen, Adrianna Vlachos, Marie-françoise O'donohue, Milena Preti, Stella M Davies, Antonis Kattamis, Michael Landowski, Christopher BurosAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth ribosomal protein regulating p53 activity that is linked to DBA.
Carol A. Mercer - One of the best experts on this subject based on the ideXlab platform.
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abstract 2024 c myc driven b cell lymphomas role of the rpl5 RPL11 5s rrna mdm2 inhibitory complex l5 l11 5s mic
Cancer Research, 2015Co-Authors: Suresh Peddigari, Carol A. Mercer, Sara C. Kozma, George ThomasAbstract:We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p53-/- and p53 +/+ Eμ-Myc cells, likely due to the inhibition of global translation. These findings demonstrate a differential abrogation of ribosome biogenesis by partial depletion of two essential RPs, which assemble at a very similar stage in 60S ribosome. For RPL7a, it led to L5/L11/5S-MIC induced p53 mediated cell death, and this was not the case for RPL11 as it is an essential component of the complex. Citation Format: Suresh Peddigari, Carol Mercer, Sara Kozma, George Thomas. c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2015-2024
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Abstract 2024: c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC)
Molecular and Cellular Biology, 2015Co-Authors: Suresh Peddigari, Carol A. Mercer, Sara C. Kozma, George ThomasAbstract:We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p53-/- and p53 +/+ Eμ-Myc cells, likely due to the inhibition of global translation. These findings demonstrate a differential abrogation of ribosome biogenesis by partial depletion of two essential RPs, which assemble at a very similar stage in 60S ribosome. For RPL7a, it led to L5/L11/5S-MIC induced p53 mediated cell death, and this was not the case for RPL11 as it is an essential component of the complex. Citation Format: Suresh Peddigari, Carol Mercer, Sara Kozma, George Thomas. c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2015-2024
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loss of tumor suppressor rpl5 RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity
Molecular and Cellular Biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
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Loss of tumor suppressor RPL5/RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity.
Molecular and cellular biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
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5s ribosomal rna is an essential component of a nascent ribosomal precursor complex that regulates the hdm2 p53 checkpoint
Cell Reports, 2013Co-Authors: Giulio Donati, Suresh Peddigari, Carol A. Mercer, George ThomasAbstract:Recently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.
Adrianna Vlachos - One of the best experts on this subject based on the ideXlab platform.
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novel deletion of rpl15 identified by array comparative genomic hybridization in diamond blackfan anemia
Human Genetics, 2013Co-Authors: Michael Landowski, Adrianna Vlachos, Mariefrancoise Odonohue, Colin A Sieff, Christopher Buros, Roxanne Ghazvinian, Nathalie MontellehryAbstract:Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30-50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60-65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.
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frameshift mutation in p53 regulator rpl26 is associated with multiple physical abnormalities and a specific pre ribosomal rna processing defect in diamond blackfan anemia
Human Mutation, 2012Co-Authors: Mee Rie Sheen, Adrianna Vlachos, Mariefrancoise Odonohue, Hanna T Gazda, Milena Preti, Stella M Davies, Antonis KattamisAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ~30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth ribosomal protein regulating p53 activity that is linked to DBA.
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frameshift mutation in p53 regulator rpl26 is associated with multiple physical abnormalities and a specific pre ribosomal rna processing defect in diamond blackfan anemia
Human Mutation, 2012Co-Authors: Adrianna Vlachos, Mariefrancoise Odonohue, Hanna T Gazda, Milena Preti, Stella M Davies, Mee Rie Shee, Antonis KattamisAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA.
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Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-rRNA processing defect in Diamond-Blackfan anemia.
Human Mutation, 2012Co-Authors: Hanna T Gazda, Leana Doherty, Mee Rie Sheen, Adrianna Vlachos, Marie-françoise O'donohue, Milena Preti, Stella M Davies, Antonis Kattamis, Michael Landowski, Christopher BurosAbstract:Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth ribosomal protein regulating p53 activity that is linked to DBA.
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ribosomal protein genes rps10 and rps26 are commonly mutated in diamond blackfan anemia
American Journal of Human Genetics, 2010Co-Authors: Leana Doherty, Mee Rie Sheen, Adrianna Vlachos, Valerie Choesmel, Mariefrancoise Odonohue, Catherine Clinton, Hal E SchneiderAbstract:Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.
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loss of tumor suppressor rpl5 RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity
Molecular and Cellular Biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
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Loss of tumor suppressor RPL5/RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity.
Molecular and cellular biology, 2013Co-Authors: Teng Teng, Carol A. Mercer, George Thomas, Philip Hexley, Stefano FumagalliAbstract:Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.
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Suprainduction of p53 by disruption of 40S and 60S ribosome biogenesis leads to the activation of a novel G2/M checkpoint
Genes & development, 2012Co-Authors: Stefano Fumagalli, Teng Teng, Vasily V. Ivanenkov, George ThomasAbstract:Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.
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Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an RPL11-translation-dependent mechanism of p53 induction
Nature cell biology, 2009Co-Authors: Stefano Fumagalli, Alessandro Di Cara, Arti Neb-gulati, Francois Natt, Sandy Schwemberger, Jonathan Hall, George F. Babcock, Rosa Bernardi, Pier Paolo Pandolfi, George ThomasAbstract:Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by RPL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5'-transcriptional start site (5'-TOP mRNAs), including that encoding RPL11, on impairment of 40S ribosome biogenesis. Increased 5'-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins, specific targeting of RPL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction.
