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

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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The 5' untranslated region of mRNA for ribosomal protein S19 is involved in its translational regulation during Xenopus development.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.816 ◽

1990 ◽

Vol 10 (2) ◽

pp. 816-822 ◽

Cited By ~ 57

Author(s):

P Mariottini ◽

F Amaldi

Keyword(s):

Ribosomal Protein ◽

Untranslated Region ◽

Ribosomal Proteins ◽

Translational Regulation ◽

Xenopus Development ◽

Untranslated Sequence ◽

Gene Coding ◽

Ribosomal Protein S19 ◽

Fused Gene

During Xenopus development, the synthesis of ribosomal proteins is regulated at the translational level. To identify the region of the ribosomal protein mRNAs responsible for their typical translational behavior, we constructed a fused gene in which the upstream sequences (promoter) and the 5' untranslated sequence (first exon) of the gene coding for Xenopus ribosomal protein S19 were joined to the coding portion of the procaryotic chloramphenicol acetyltransferase (CAT) gene deleted of its own 5' untranslated region. This fused gene was introduced in vivo by microinjection into Xenopus fertilized eggs, and its activity was monitored during embryogenesis. By analyzing the pattern of appearance of CAT activity and the distribution of the S19-CAT mRNA between polysomes and messenger ribonucleoproteins, it was concluded that the 35-nucleotide-long 5' untranslated region of the S19 mRNA is able to confer to the fused S19-CAT mRNA the translational behavior typical of ribosomal proteins during Xenopus embryo development.

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Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5281-5288.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5281-5288

Author(s):

W M Wormington

Keyword(s):

Ribosomal Protein ◽

Mammalian Cells ◽

Ribosomal Proteins ◽

5S Rrna ◽

Yeast Cells ◽

Developmental Expression ◽

Rrna Synthesis ◽

Ribosome Assembly ◽

Subunit Assembly ◽

Ribosomal Protein L5

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

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A DEAD-box protein regulates ribosome assembly through control of ribosomal protein synthesis

Nucleic Acids Research ◽

10.1093/nar/gkz502 ◽

2019 ◽

Vol 47 (15) ◽

pp. 8193-8206 ◽

Cited By ~ 2

Author(s):

Isabelle Iost ◽

Chaitanya Jain

Keyword(s):

Protein Synthesis ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Large Family ◽

Ribosome Assembly ◽

Protein Abundance ◽

Ribosomal Assembly ◽

Dead Box ◽

Ribosomal Protein Synthesis ◽

Intrinsic Transcription Termination

Abstract DEAD-box proteins (DBPs) comprise a large family of proteins that most commonly have been identified as regulators of ribosome assembly. The Escherichia coli DBP, SrmB, represents a model bacterial DBP whose absence impairs formation of the large ribosomal subunit (LSU). To define the basis for SrmB function, suppressors of the ribosomal defect of ΔsrmB strains were isolated. The major class of suppressors was found to map to the 5′ untranslated region (UTR) of the rplM-rpsI operon, which encodes the ribosomal proteins (r-proteins) L13 and S9. An analysis of protein abundance indicated that both r-proteins are under-produced in the ΔsrmB strain, but are increased in these suppressors, implicating r-protein underproduction as the molecular basis for the observed ribosomal defects. Reduced r-protein synthesis was determined to be caused by intrinsic transcription termination within the rplM 5′ UTR that is abrogated by SrmB. These results reveal a specific mechanism for DBP regulation of ribosomal assembly, indirectly mediated through its effects on r-protein expression.

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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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Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5281 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5281-5288 ◽

Cited By ~ 35

Author(s):

W M Wormington

Keyword(s):

Ribosomal Protein ◽

Mammalian Cells ◽

Ribosomal Proteins ◽

5S Rrna ◽

Yeast Cells ◽

Developmental Expression ◽

Rrna Synthesis ◽

Ribosome Assembly ◽

Subunit Assembly ◽

Ribosomal Protein L5

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

Blood ◽

10.1182/blood.v118.21.3432.3432 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3432-3432

Author(s):

Hong-Yan Du ◽

M. Tarek Elghetany ◽

Blanche P Alter ◽

Akiko Shimamura

Keyword(s):

Bone Marrow ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Mrna Levels ◽

Protein Levels ◽

Diamond Blackfan Anemia ◽

Human Cd34 ◽

Cd34 Cells ◽

P53 Activation ◽

Ribosomal Stress

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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Structure and expression of ribosomal protein genes in Xenopus laevis

Biochemistry and Cell Biology ◽

10.1139/o95-104 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 969-977 ◽

Cited By ~ 16

Author(s):

Francesco Amaldi ◽

Olga Camacho-Vanegas ◽

Francesco Cecconi ◽

Fabrizio Loreni ◽

Beatrice Cardinali ◽

...

Keyword(s):

Xenopus Laevis ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Translational Regulation ◽

Cultured Cells ◽

Structural Features ◽

Ribosomal Protein Genes ◽

Translation Apparatus ◽

And Function

In Xenopus laevis, as well as in other vertebrates, ribosomal proteins (r-proteins) are coded by a class of genes that share some organizational and structural features. One of these, also common to genes coding for other proteins involved in the translation apparatus synthesis and function, is the presence within their introns of sequences coding for small nucleolar RNAs. Another feature is the presence of common structures, mainly in the regions surrounding the 5′ ends, involved in their coregulated expression. This is attained at various regulatory levels: transcriptional, posttranscriptional, and translational. Particular attention is given here to regulation at the translational level, which has been studied during Xenopus oogenesis and embryogenesis and also during nutritional changes of Xenopus cultured cells. This regulation, which responds to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA (ribosomal protein mRNA) engaged on polysomes. A typical 5′ untranslated region characterizing all vertebrate rp-mRNAs analyzed to date is responsible for this translational behaviour: it is always short and starts with an 8–12 nucleotide polypyrimidine tract. This region binds in vitro some proteins that can represent putative trans-acting factors for this translational regulation.Key words: ribosomal proteins, snoRNA, translational regulation, Xenopus laevis.

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SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Journal of Bacteriology ◽

10.1128/jb.181.10.3136-3143.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3136-3143 ◽

Cited By ~ 31

Author(s):

Nelson Lopez ◽

John Halladay ◽

William Walter ◽

Elizabeth A. Craig

Keyword(s):

Heat Shock ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Chain Complex ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Genes Encoding ◽

Synthetic Capacity ◽

Translational Apparatus ◽

Temperature Upshift

ABSTRACT Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock theEXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

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Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

Scientific Reports ◽

10.1038/s41598-020-76239-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yasuko Matsuki ◽

Yoshitaka Matsuo ◽

Yu Nakano ◽

Shintaro Iwasaki ◽

Hideyuki Yoko ◽

...

Keyword(s):

Er Stress ◽

Ribosomal Protein ◽

Translational Control ◽

Ribosomal Proteins ◽

Translational Regulation ◽

Mrna Translation ◽

Ribosome Profiling ◽

Independent Manner ◽

Eif2α Phosphorylation ◽

The Unfolded Protein Response

AbstracteIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

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Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.2.10.1199 ◽

1982 ◽

Vol 2 (10) ◽

pp. 1199-1204 ◽

Cited By ~ 11

Author(s):

E Kraig ◽

J E Haber ◽

M Rosbash

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Relative Rate ◽

Synthesis Rate ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Mrna Metabolism

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.

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