A memory of eS25 loss drives resistance phenotypes

ABSTRACTIn order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

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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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Ribosomal protein gene deletions in Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-08-375170 ◽

2011 ◽

Vol 118 (26) ◽

pp. 6943-6951 ◽

Cited By ~ 87

Author(s):

Jason E. Farrar ◽

Adrianna Vlachos ◽

Eva Atsidaftos ◽

Hannah Carlson-Donohoe ◽

Thomas C. Markello ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Protein Gene ◽

Single Nucleotide Polymorphism Array ◽

Ribosomal Protein Gene ◽

Small Subunit ◽

Ribosomal Protein Genes ◽

Gene Deletions ◽

Diamond Blackfan Anemia ◽

Genome Wide

Abstract Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

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Constitutive transcription of yeast ribosomal protein gene TCM1 is promoted by uncommon cis- and trans-acting elements.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4328 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4328-4341 ◽

Cited By ~ 35

Author(s):

K G Hamil ◽

H G Nam ◽

H M Fried

Keyword(s):

Ribosomal Protein ◽

Protein Gene ◽

Biochemical Characterization ◽

Transcriptional Activator ◽

Ribosomal Protein Gene ◽

Cross Linking ◽

Ribosomal Protein Genes ◽

Cis Acting ◽

Transcriptional Activator Protein

The DNA sequence UAST (TCGTTTTGTACGTTTTTCA) was found to mediate transcription of yeast ribosomal protein gene TCM1. UAST was defined as a transcriptional activator on the basis of loss of transcription accompanying deletions of all or part of UAST, orientation-independent restoration of transcription promoted by a synthetic UAST oligomer inserted either into TCM1 or into the yeast CYC1 gene lacking its transcriptional activation region, and diminished transcription following nucleotide alterations in UAST. UAST bound in vitro to a protein denoted TAF (TCM1 activation factor); TAF was concluded to be a transcriptional activator protein because nucleotide alterations in UAST that diminished transcription in vivo also diminished TAF binding in vitro. The sequence of UAST bore no obvious resemblance to UASrpg, the principal cis-acting element common to most yeast ribosomal protein genes. Likewise, TAF was distinguished from the UASrpg-binding protein TUF, since (i) TAF and TUF were chromatographically separable, (ii) binding of either TAF or TUF to its corresponding UAS was unaffected by an excess of UASrpg or UAST DNA, respectively, and (iii) photochemical cross-linking experiments showed that TAF was a protein of 147 kilodaltons (kDa), while TUF was detected as an approximately 120-kDa polypeptide, consistent with its known size. Cross-linking experiments also revealed that both UAST and UASrpg bound a second heretofore unobserved 82-kDa protein; binding of this additional protein appeared to require binding of TAF or TUF. On the basis of the biochemical characterization of TAF and a lack of sequence similarity between UAST and UASrpg, we suggest that transcription of TCM1 is mediated by a cis-acting sequence and at least one trans-acting factor different from the elements which promote transcription of most other ribosomal protein genes. A second trans-acting factor may be shared by TCM1 and other ribosomal protein genes; this factor could mediate coordinate regulation of these genes.

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Coexpression of Escherichia coli obgE, Encoding the Evolutionarily Conserved Obg GTPase, with Ribosomal Proteins L21 and L27

Journal of Bacteriology ◽

10.1128/jb.00159-16 ◽

2016 ◽

Vol 198 (13) ◽

pp. 1857-1867 ◽

Cited By ~ 1

Author(s):

Rim Maouche ◽

Hector L. Burgos ◽

Laetitia My ◽

Julie P. Viala ◽

Richard L. Gourse ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Transcription Initiation ◽

Ribosomal Proteins ◽

Ribosomal Gene ◽

Small Gtpases ◽

Ribosomal Protein Genes ◽

Transcriptional Terminator ◽

Content Type

ABSTRACTMultiple essential small GTPases are involved in the assembly of the ribosome or in the control of its activity. Among them, ObgE (CgtA) has been shown recently to act as a ribosome antiassociation factor that binds to ppGpp, a regulator whose best-known target is RNA polymerase. The present study was aimed at elucidating the expression ofobgEinEscherichia coli. We show thatobgEis cotranscribed with ribosomal protein genesrplUandrpmAand with a gene of unknown function,yhbE. We show here that about 75% of the transcripts terminate beforeobgE, because there is a transcriptional terminator betweenrpmAandyhbE. As expected for ribosomal protein operons, expression was highest during exponential growth, decreased during entry into stationary phase, and became almost undetectable thereafter. Expression of the operon was derepressed in mutants lacking ppGpp or DksA. However, regulation by these factors appears to occur post-transcription initiation, since no effects of ppGpp and DksA onrplUpromoter activity were observedin vitro.IMPORTANCEThe conserved and essential ObgE GTPase binds to the ribosome and affects its assembly. ObgE has also been reported to impact chromosome segregation, cell division, resistance to DNA damage, and, perhaps most interestingly, persister formation and antibiotic tolerance. However, it is unclear whether these effects are related to its role in ribosome formation. Despite its importance, no studies on ObgE expression have been reported. We demonstrate here thatobgEis expressed from an operon encoding two ribosomal proteins, that the operon's expression varies with the growth phase, and that it is dependent on the transcription regulators ppGpp and DksA. Our results thus demonstrate thatobgEexpression is coupled to ribosomal gene expression.

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Altered Cellular Responses by Varying Expression of a Ribosomal Protein Gene: Sequential Coordination of Enhancement and Suppression of Ribosomal Protein S3a Gene Expression Induces Apoptosis

The Journal of Cell Biology ◽

10.1083/jcb.141.3.741 ◽

1998 ◽

Vol 141 (3) ◽

pp. 741-753 ◽

Cited By ~ 110

Author(s):

Honami Naora ◽

Izumi Takai ◽

Masakazu Adachi ◽

Hiroto Naora

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Chromatin Condensation ◽

Ribosomal Protein Gene ◽

Cellular Responses ◽

Cell Contact ◽

Morphological Transformation ◽

Cell Shrinkage ◽

Nih 3T3

A growing body of evidence indicates that individual ribosomal proteins and changes in their expression, participate in, and modulate, a variety of cellular activities. Our earlier studies have found that apoptosis could be induced by inhibiting expression of ribosomal protein S3a (RPS3a) in many tumor cells which constitutively express RPS3a at levels much higher than in normal cells. This study aimed to investigate cellular responses to enhancement of RPS3a expression, and whether apoptosis could be induced by sequential alterations in RPS3a expression involving enhancement from an initially low constitutive level, followed by suppression. Stably transfected NIH 3T3– derived cell lines were established in which exogenous RPS3a expression could be readily manipulated. Enhancement of RPS3a expression appeared to induce transformation as assessed by well-established criteria such as foci formation and anchorage-independent growth in vitro, and formation of tumors in nude mice. These properties were compared with those observed in ras-transformed NIH 3T3 cells. Apparent transformation occurred only when enhanced RPS3a-expressing cells were in close cell–cell contact. Suppression of enhanced RPS3a expression was observed to induce apoptosis as assessed by various morphological and biochemical characteristics including cell shrinkage, membrane blebbing, chromatin condensation, nuclear and cell fragmentation, phosphatidylserine externalization, and internucleosomal DNA fragmentation. This induction of apoptosis was not specific to apparently transformed cells, as cells at low confluence, which likewise expressed RPS3a at enhanced levels but exhibited no morphological transformation, underwent apoptosis when RPS3a expression was inhibited. These results support a role for RPS3a in the apoptotic process, but not as an oncoprotein per se.

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Constitutive transcription of yeast ribosomal protein gene TCM1 is promoted by uncommon cis- and trans-acting elements

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4328-4341.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4328-4341

Author(s):

K G Hamil ◽

H G Nam ◽

H M Fried

Keyword(s):

Ribosomal Protein ◽

Protein Gene ◽

Biochemical Characterization ◽

Transcriptional Activator ◽

Ribosomal Protein Gene ◽

Cross Linking ◽

Ribosomal Protein Genes ◽

Cis Acting ◽

Transcriptional Activator Protein

The DNA sequence UAST (TCGTTTTGTACGTTTTTCA) was found to mediate transcription of yeast ribosomal protein gene TCM1. UAST was defined as a transcriptional activator on the basis of loss of transcription accompanying deletions of all or part of UAST, orientation-independent restoration of transcription promoted by a synthetic UAST oligomer inserted either into TCM1 or into the yeast CYC1 gene lacking its transcriptional activation region, and diminished transcription following nucleotide alterations in UAST. UAST bound in vitro to a protein denoted TAF (TCM1 activation factor); TAF was concluded to be a transcriptional activator protein because nucleotide alterations in UAST that diminished transcription in vivo also diminished TAF binding in vitro. The sequence of UAST bore no obvious resemblance to UASrpg, the principal cis-acting element common to most yeast ribosomal protein genes. Likewise, TAF was distinguished from the UASrpg-binding protein TUF, since (i) TAF and TUF were chromatographically separable, (ii) binding of either TAF or TUF to its corresponding UAS was unaffected by an excess of UASrpg or UAST DNA, respectively, and (iii) photochemical cross-linking experiments showed that TAF was a protein of 147 kilodaltons (kDa), while TUF was detected as an approximately 120-kDa polypeptide, consistent with its known size. Cross-linking experiments also revealed that both UAST and UASrpg bound a second heretofore unobserved 82-kDa protein; binding of this additional protein appeared to require binding of TAF or TUF. On the basis of the biochemical characterization of TAF and a lack of sequence similarity between UAST and UASrpg, we suggest that transcription of TCM1 is mediated by a cis-acting sequence and at least one trans-acting factor different from the elements which promote transcription of most other ribosomal protein genes. A second trans-acting factor may be shared by TCM1 and other ribosomal protein genes; this factor could mediate coordinate regulation of these genes.

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Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

Molecular and Cellular Biology ◽

10.1128/mcb.3.3.457-465.1983 ◽

1983 ◽

Vol 3 (3) ◽

pp. 457-465

Author(s):

C H Kim ◽

J R Warner

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Temperature Shift ◽

Restrictive Temperature ◽

Ribosomal Protein Genes ◽

Wild Type ◽

Temperature Shock ◽

Mild Temperature ◽

Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

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The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein.

Genetics ◽

10.1093/genetics/132.2.375 ◽

1992 ◽

Vol 132 (2) ◽

pp. 375-386 ◽

Cited By ~ 1

Author(s):

A Vincent ◽

S W Liebman

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Dna Sequences ◽

Ribosomal Proteins ◽

Amino Acid Sequences ◽

Ribosomal Protein Genes ◽

Significant Homology ◽

A Cell ◽

Functional Equivalent ◽

Ribosomal Protein S13

Abstract The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

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Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2723-2735.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2723-2735 ◽

Cited By ~ 1

Author(s):

C M Moehle ◽

A G Hinnebusch

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Ribosomal Protein ◽

Binding Sites ◽

Protein Gene ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Biosynthetic Genes ◽

Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

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