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

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

10.1101/2020.09.24.311365 ◽

2020 ◽

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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Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα

Molecular Biology of the Cell ◽

10.1091/mbc.e14-02-0704 ◽

2014 ◽

Vol 25 (10) ◽

pp. 1686-1697 ◽

Cited By ~ 60

Author(s):

Thomas D. Baird ◽

Lakshmi Reddy Palam ◽

Michael E. Fusakio ◽

Jeffrey A. Willy ◽

Christopher M. Davis ◽

...

Keyword(s):

Er Stress ◽

Cell Fate ◽

Translational Control ◽

Translational Regulation ◽

Cultured Cells ◽

Mrna Translation ◽

Open Reading Frames ◽

Α Subunit ◽

A Genome ◽

The Unfolded Protein Response

Disruption of protein folding in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PKR-like ER kinase (PERK/EIF2AK3) phosphorylation of the α subunit of eIF2 (eIF2α∼P), which represses global translation coincident with preferential translation of mRNAs, such as activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP), that serve to implement UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary over a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α∼P, whereas a notable cohort of key regulators are subject to preferential translation. From the latter group, we identified the α isoform of inhibitor of Bruton's tyrosine kinase (IBTKα) as being subject to both translational and transcriptional induction during eIF2α∼P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves stress-induced relief of two inhibitory upstream open reading frames in the 5′-leader of the transcript. Depletion of IBTKα by short hairpin RNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKα is a key regulator in determining cell fate during the UPR.

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IRE1α-Dependent Decay of CReP/Ppp1r15b mRNA Increases Eukaryotic Initiation Factor 2α Phosphorylation and Suppresses Protein Synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.00215-15 ◽

2015 ◽

Vol 35 (16) ◽

pp. 2761-2770 ◽

Cited By ~ 16

Author(s):

Jae-Seon So ◽

Sungyun Cho ◽

Sang-Hyun Min ◽

Scot R. Kimball ◽

Ann-Hwee Lee

Keyword(s):

Protein Synthesis ◽

Er Stress ◽

Translational Control ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Regulatory Subunit ◽

Dependent Manner ◽

Eukaryotic Translation ◽

Eif2α Phosphorylation ◽

The Unfolded Protein Response

The unfolded protein response (UPR) regulates endoplasmic reticulum (ER) homeostasis and protects cells from ER stress. IRE1α is a central regulator of the UPR that activates the transcription factor XBP1s through an unconventional splicing mechanism using its endoribonuclease activity. IRE1α also cleaves certain mRNAs containing XBP1-like secondary structures to promote the degradation of these mRNAs, a process known as regulated IRE1α-dependent decay (RIDD). We show here that the mRNA of CReP/Ppp1r15b, a regulatory subunit of eukaryotic translation initiation factor 2α (eIF2α) phosphatase, is a RIDD substrate. eIF2α plays a central role in the integrated stress response by mediating the translational attenuation to decrease the stress level in the cell. CReP expression was markedly suppressed in XBP1-deficient mice livers due to hyperactivated IRE1α. Decreased CReP expression caused the induction of eIF2α phosphorylation and the attenuation of protein synthesis in XBP1-deficient livers. ER stress also suppressed CReP expression in an IRE1α-dependent manner, which increased eIF2α phosphorylation and consequently attenuated protein synthesis. Taken together, the results of our study reveal a novel function of IRE1α in the regulation of eIF2α phosphorylation and the translational control.

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Glucocorticoids selectively inhibit translation of ribosomal protein mRNAs in P1798 lymphosarcoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2691-2699.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2691-2699 ◽

Cited By ~ 1

Author(s):

O Meyuhas ◽

E A Thompson ◽

R P Perry

Keyword(s):

Ribosomal Protein ◽

Translational Control ◽

Ribosomal Proteins ◽

Mrna Translation ◽

Substantial Decrease ◽

Rrna Synthesis ◽

Dexamethasone Treatment ◽

Selective Suppression

When P1798 murine lymphosarcoma cells are exposed to 10(-7) M dexamethasone, there is a dramatic inhibition of rRNA synthesis, which is completely reversible when the hormone is withdrawn. In the present experiments we examined whether dexamethasone treatment causes any alteration in the accumulation or utilization of mRNAs that encode ribosomal proteins (rp mRNAs). No effect on the accumulation of six different rp mRNAs was detected. However, the translation of five of six rp mRNAs was selectively inhibited in the presence of the hormone, as judged by a substantial decrease in ribosomal loading. Normal translation of rp mRNA was resumed within a few hours after hormone withdrawal. In untreated or fully recovered cells, the distribution of rp mRNAs between polyribosomes and free ribonucleoprotein is distinctly bimodal, suggesting that rp mRNAs are subject to a particular form of translational control in which they are either translationally inactive or fully loaded with ribosomes. A possible relationship between this mode of translational control and the selective suppression of rp mRNA translation by glucocorticoids is discussed.

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Glucocorticoids selectively inhibit translation of ribosomal protein mRNAs in P1798 lymphosarcoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2691 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2691-2699 ◽

Cited By ~ 86

Author(s):

O Meyuhas ◽

E A Thompson ◽

R P Perry

Keyword(s):

Ribosomal Protein ◽

Translational Control ◽

Ribosomal Proteins ◽

Mrna Translation ◽

Substantial Decrease ◽

Rrna Synthesis ◽

Dexamethasone Treatment ◽

Selective Suppression

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Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

Scientific Reports ◽

10.1038/s41598-020-79379-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kyle A. Cottrell ◽

Ryan C. Chiou ◽

Jason D. Weber

Keyword(s):

Protein Synthesis ◽

Tumor Cells ◽

Ribosomal Proteins ◽

Human Cancer ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Gene Encoding ◽

Terminal Oligopyrimidine ◽

Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

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A UV-Induced Mutation in Neurospora That Affects Translational Regulation in Response to Arginine

Genetics ◽

10.1093/genetics/142.1.117 ◽

1996 ◽

Vol 142 (1) ◽

pp. 117-127 ◽

Cited By ~ 3

Author(s):

Michael Freitag ◽

Nelima Dighde ◽

Matthew S Sachs

Keyword(s):

Translational Control ◽

Translational Regulation ◽

Fusion Gene ◽

Mrna Translation ◽

Small Subunit ◽

Upstream Open Reading Frame ◽

Control Mechanisms ◽

Carbamoyl Phosphate ◽

Altered Expression ◽

Reading Frame

The Neurospora crmsu arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript’s 5′ region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and urg-2 genes; the other, altered expression of urg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5′ region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFS suggest common elements in translational control mechanisms.

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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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ARF suppresses 5’-terminal oligopyrimidine mRNA translation

10.1101/2020.02.07.939165 ◽

2020 ◽

Author(s):

Kyle A. Cottrell ◽

Ryan C. Chiou ◽

Jason D. Weber

Keyword(s):

Protein Synthesis ◽

Tumor Cells ◽

Ribosomal Proteins ◽

Human Cancer ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Gene Encoding ◽

Terminal Oligopyrimidine ◽

Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How many of the mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5’-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 caused a similar increase in 5’-TOP mRNA translation. The 5’-TOP regulators mTORC1, eIF4G1 and LARP1 are dysregulated in ARF and p53 null cells.

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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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