P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.

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Acquired ribosomopathies in leukemia and solid tumors

Hematology ◽

10.1182/asheducation-2017.1.716 ◽

2017 ◽

Vol 2017 (1) ◽

pp. 716-719 ◽

Cited By ~ 6

Author(s):

Adrianna Vlachos

Keyword(s):

Bone Marrow ◽

Ribosomal Protein ◽

Solid Tumors ◽

Ribosomal Proteins ◽

Bone Marrow Failure ◽

Ribosomal Protein Gene ◽

Small Subunit ◽

Gene Encoding ◽

Bone Marrow Failure Syndrome ◽

Diamond Blackfan Anemia

AbstractA mutation in the gene encoding the small subunit-associated ribosomal protein RPS19, leading to RPS19 haploinsufficiency, is one of the ribosomal protein gene defects responsible for the rare inherited bone marrow failure syndrome Diamond Blackfan anemia (DBA). Additional inherited and acquired defects in ribosomal proteins (RPs) continue to be identified and are the basis for a new class of diseases called the ribosomopathies. Acquired RPS14 haploinsufficiency has been found to be causative of the bone marrow failure found in 5q– myelodysplastic syndromes. Both under- and overexpression of RPs have also been implicated in several malignancies. This review will describe the somatic ribosomopathies that have been found to be associated with a variety of solid tumors as well as leukemia and will review cancers in which over- or underexpression of these proteins seem to be associated with outcome.

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Inhibition of Splicing Factor SF3B1: Evaluating Effects on Iron Metabolism in K562 and CD34+ Cells

Blood ◽

10.1182/blood.v124.21.1348.1348 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1348-1348

Author(s):

Simon Knight ◽

Robert K. Bressin ◽

Upamanyu Basu ◽

Kazunori Koide ◽

Andrew Dancis

Keyword(s):

Ribosomal Protein ◽

Iron Metabolism ◽

Iron Uptake ◽

K562 Cells ◽

Mrna Levels ◽

Splice Form ◽

Exon 2 ◽

Protein Levels ◽

Cellular Iron ◽

Cd34 Cells

Abstract Somatic mutations in the RNA splicing factor SF3B1 have been found in hematopoietic cells from patients with myelodysplastic syndrome. The association is particularly strong for patients with anemia with ringed sideroblasts, where 65 - 75 % show alterations in SF3B1. Ringed sideroblasts are erythroid precursors with insoluble iron aggregates in mitochondria and deficient hemoglobin synthesis. Thus these cells demonstrate a defect in iron metabolism. SF3B1 is a core component of the U2 snRNP spliceosome, responsible for the majority of mRNA maturation, raising the question of how alteration of SF3B1 might perturb cellular iron metabolism. Meayamycin B, a synthetic analog of the natural product FR901464, is an inhibitor of SF3B1, and here we describe the effects of this compound on cellular iron metabolism in the K562 leukemic cell line and in CD34+ cells isolated from cord blood. Inhibition of SF3B1 by meayamycin B was confirmed by analysis of spliced forms of Mcl-1 mRNA, a known target of SF3B1. In untreated cells, a long form (Mcl-lL) arises from inclusion of exon 2 in the final mRNA. In treated cells, inhibition of SF3B1 splicing generates a short form (Mcl-lS) that lacks exon 2. In the presence of 3 to 10 nM meayamycin B the short splice-form (Mcl-1S) became the dominant species. To examine the effect of inhibition of SF3B1 on iron metabolism, K562 cells were exposed to meayamycin B for 24 hours, with controls of 50 µM hemin and 50 µM deferoxamine (DFO). Steady state mRNA levels of transferrin receptor (TFR-1), ABCB7 (mutations in which are associated with inherited sideroblastic anemia and ataxia), and NDRG1 (n-myc downstream regulated gene, previously shown to be induced by DFO) were significantly down regulated by meayamycin B, as determined by qRT-PCR. NDUFS8 mRNA that encodes a subunit of mitochondrial complex 1 and has previously been shown to be iron-responsive, did not change in response to meayamycin B, hemin, or DFO. However, MRLP19 that encodes a mitochondrial ribosomal component, was strongly down-regulated by meayamycin B. In contrast, cytosolic ribosomal protein RPLP0 was not affected by meayamycin B, hemin or DFO and was used as the endogenous control. Analysis of protein levels by western blotting indicated that meayamycin B increased levels of ferritin (heavy chain) and decreased TFR-1. In concordance with the decreased TFR-1 protein levels, meayamycin B treated K562 cells exhibited significant downregulation of iron uptake from 55Fe-transferrin, similar to that observed for hemin treated cells. DFO treatment significantly increased transferrin iron uptake, compared to untreated controls. A similar set of experiments was performed with CD34+ cells, before or after differentiation with erythropoietin. Meayamycin was efficacious in these cells, as shown by disappearance of the long splice-form Mcl-lL and appearance of the short splice form Mcl-1S in cells exposed to the compound for 16 h. TFR-1 mRNA exhibited a dose dependent decrease in response to meayamycin (both pre and post epo treatment). Neither hemin nor DFO altered TFR-1 mRNA levels, or protein levels. In non-epo treated CD34+ cells, exposure to meayamycin caused a small decrease in ferritin protein levels, but ferritin protein increased in epo-treated cells exposed to meayamycin. Hemin increased ferritin protein in both non-epo and epo treated cells. Mitochondrial ribosomal protein MRPL19 RNA was significantly decreased by meayamycin in all cases, but cytoplasmic ribosomal protein RPLP0 was unaffected by meayamycin. Ribosomal protein levels were not responsive to iron manipulations with hemin or DFO. To summarize, treatment of cells with meayamycin B inhibits SF3B1 function in K562 cells or CD34+ cells. The effects are quicker and cleaner than siRNA, making possible closer functional correlations. Meayamycin treated K562 cells exhibited downregulation of TFR-1 and TFR-1 iron uptake, as if they were sensing high cellular iron levels. Note that this is the opposite of the expected result, because iron loading in the setting of sideroblastic anemia would be expected to be associated with increased cellular iron uptake. In CD34+ cells, a similar downregulation of TFR-1 levels was observed, although iron uptake has not yet been directly measured. The strong decrease in mitochondrial ribosomal protein expression indicates a specific mitochondrial effect of SF3B1 inhibition, which may be an important mediator of iron metabolism. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic Mechanism in Diamond-Blackfan Anemia: Late Erythroid Development Is Not Affected by Ribosomal Protein S19 Deficiency.

Blood ◽

10.1182/blood.v104.11.719.719 ◽

2004 ◽

Vol 104 (11) ◽

pp. 719-719

Author(s):

Johan Flygare ◽

Thomas Kiefer ◽

Koichi Miyake ◽

Taiju Utsugisawa ◽

Isao Hamaguchi ◽

...

Keyword(s):

Ribosomal Protein ◽

Liquid Culture ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Protein Levels ◽

Diamond Blackfan Anemia ◽

Cd34 Cells ◽

Ribosomal Protein S19 ◽

Erythroid Development ◽

In Cells

Abstract Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. It is unknown how the ribosomal protein deficiency leads to anemia. We previously developed three lentiviral vectors expressing siRNA against RPS19 and one scramble control vector. All vectors also express GFP. We have previously shown that transduction of CD34+ bone marrow (BM) cells with the siRNA vectors reduced RPS19 mRNA levels, resulting in formation of fewer erythroid colonies. In the present study, we have demonstrated downregulation of RPS19 protein in siRNA treated cells. RPS19 protein levels decreased over time and were reduced to 40-60% of normal in cells transduced with all three siRNA vectors 5 days after transduction. We asked which stage of erythroid development is most affected by RPS19 deficiency. After 7 days in liquid culture supporting erythroid differentiation Glycophorin A (GlyA) and CD71 expression was examined by FACS. RPS19-silenced as well as DBA patient CD34+ cells exhibited a block in erythroid differentiation seen as an increased fraction of GlyAneg CD71low cells while the fractions of CD71high GlyAintermediate and GlyAhigh cells decreased. We cultured untransduced CD34+ cells in liquid culture for 7 days and isolated CD71high GlyA intermediate cells that are highly enriched in CFU-E and do not contain BFU-E. These cells were transduced with RPS19 siRNA vectors. Further erythroid maturation from CFU-E (CD71high GlyAintermediate) to more mature erythroid cells (GlyAhigh) was not affected by RPS19 silencing suggesting that the main block in erythroid development occurs prior to the CFU-E formation. The failure of erythroid differentiation correlated with the decrease in RPS19 protein levels. Transduction with a lentivirus expressing an siRNA-resistant RPS19 transcript rescued both the erythroid progenitor proliferation and differentiation, showing that the DBA-like phenotype is specific to the RPS19 deficiency. Finally we cultured the cells in liquid medium supporting both erythroid and myeloid differentiation. Proliferation decreased while the ratio of mature myeloid to erythroid cells increased 3 fold in cells transduced with the 2 most efficient siRNA-vectors, meaning that erythroid development is more sensitive to low RPS19 levels than myeloid development. When RPS19 is downregulated to intermediate levels, erythroid differentiation and proliferation of erythroid progenitors is severely reduced. More severe reduction of RPS19 impairs proliferation of myeloid progenitors as well, leading to a reduced output of myeloid progeny. Although our data cannot rule out hypothetical extraribosomal mechanisms we suggest that the major clinical findings in RPS19 deficient DBA can be explained by insufficient protein translation. This study shows for the first time that RPS19 protein downregulation decreases the proliferative capacity of hematopoietic progenitors leading to an early defect in erythroid development.

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L-leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway

Blood ◽

10.1182/blood-2011-10-382986 ◽

2012 ◽

Vol 120 (11) ◽

pp. 2214-2224 ◽

Cited By ~ 94

Author(s):

Elspeth M. Payne ◽

Maria Virgilio ◽

Anupama Narla ◽

Hong Sun ◽

Michelle Levine ◽

...

Keyword(s):

Ribosomal Proteins ◽

Mrna Translation ◽

Mtor Pathway ◽

Developmental Defects ◽

Diamond Blackfan Anemia ◽

Common Basis ◽

Human Cd34 ◽

Chromosome 5Q ◽

Cd34 Cells ◽

The Common

Abstract Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34+ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

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Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1799 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1799-1813 ◽

Cited By ~ 49

Author(s):

E de Beus ◽

J S Brockenbrough ◽

B Hong ◽

J P Aris

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Ribosomal Protein ◽

Nucleolar Protein ◽

Mrna Levels ◽

Multicopy Plasmid ◽

Yeast Saccharomyces Cerevisiae ◽

Protein Levels ◽

Significant Amino Acid Sequence ◽

Ribosomal Protein L3

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

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Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-08-371963 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6087-6096 ◽

Cited By ~ 82

Author(s):

Pekka Jaako ◽

Johan Flygare ◽

Karin Olsson ◽

Ronan Quere ◽

Mats Ehinger ◽

...

Keyword(s):

Bone Marrow ◽

Rna Interference ◽

Ribosomal Protein ◽

Mouse Models ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Down Regulation ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Ribosomal Protein S19

Abstract Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

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Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

10.1101/2021.10.05.463164 ◽

2021 ◽

Author(s):

Benjamin Pillet ◽

Alfonso Méndez-Godoy ◽

Guillaume Murat ◽

Sébastien Favre ◽

Michael Stumpe ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Translational Regulation ◽

De Novo ◽

Spatial Separation ◽

Ribosome Assembly ◽

Mrna Levels ◽

Ribosomal Assembly ◽

Surplus Production ◽

Regulatory Machinery

AbstractThe biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

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Targeted Disruption of the Ribosomal Protein S19 Gene Is Lethal Prior to Implantation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.4032-4037.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 4032-4037 ◽

Cited By ~ 101

Author(s):

Hans Matsson ◽

Edward J. Davey ◽

Natalia Draptchinskaia ◽

Isao Hamaguchi ◽

Andreas Ooka ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Normal Growth ◽

Lethal Effect ◽

Blastocyst Stage ◽

Targeted Disruption ◽

Gene Encoding ◽

Diamond Blackfan Anemia ◽

Associated Malformations ◽

Ribosomal Protein S19

ABSTRACT The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19 −/− do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.

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Continued functioning of the secretory pathway is essential for ribosome synthesis.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2493 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2493-2502 ◽

Cited By ~ 105

Author(s):

K Mizuta ◽

J R Warner

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Secretory Pathway ◽

Regulatory Elements ◽

Carboxypeptidase Y ◽

Temperature Sensitive ◽

Mrna Levels ◽

Nonpermissive Temperature ◽

Ribosome Synthesis ◽

Secretion Pathway

To explore the regulatory elements that maintain the balanced synthesis of the components of the ribosome, we isolated a temperature-sensitive (ts) mutant of Saccharomyces cerevisiae in which transcription both of rRNA and of ribosomal protein genes is defective at the nonpermissive temperature. Temperature sensitivity for growth is recessive and segregates 2:2. A gene that complements the ts phenotype was cloned from a genomic DNA library. Sequence analysis revealed that this gene is SLY1, encoding a protein essential for protein and vesicle transport between the endoplasmic reticulum and the Golgi apparatus. In the strain carrying our ts allele of SLY1, accumulation of the carboxypeptidase Y precursor was detected at the nonpermissive temperature, indicating that the secretory pathway is defective. To ask whether the effect of the ts allele on ribosome synthesis was specific for sly1 or was a general result of the inactivation of the secretion pathway, we assayed the levels of mRNA for several ribosomal proteins in cells carrying ts alleles of sec1, sec7, sec11, sec14, sec18, sec53, or sec63, representing all stages of secretion. In each case, the mRNA levels were severely depressed, suggesting that this is a common feature in mutants of protein secretion. For the mutants tested, transcription of rRNA was also substantially reduced. Furthermore, treatment of a sensitive strain with brefeldin A at a concentration sufficient to block the secretion pathway also led to a decrease of the level of ribosomal protein mRNA, with kinetics suggesting that the effect of a secretion defect is manifest within 15 to 30 min. We conclude that the continued function of the entire secretion pathway is essential for the maintenance of ribosome synthesis. The apparent coupling of membrane synthesis and ribosome synthesis suggest the existence of a regulatory network that connects the production of the various structural elements of the cell.

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