An integrative omics approach reveals posttranscriptional mechanisms underlying circadian temperature compensation

A defining property of circadian clocks is temperature compensation, characterized by the resilience of circadian free-running periods against changes in environmental temperature. As an underlying mechanism, the balance or critical reaction hypothesis have been proposed. While the former supposes a temperature-dependent balancing of reactions with opposite effects on circadian period, the latter assumes an insensitivity of certain critical period determining regulations upon temperature changes. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, abolishes circadian temperature compensation in U-2 OS cells. We apply a combination of 3'-End-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild type and CPSF6 knock-down cells and their dependency on temperature. Analyzing differential responses upon temperature changes in wild type and CPSF6 knockdown cells reveals candidate genes underlying circadian temperature compensation. We identify that eukaryotic translation initiation factor 2 subunit 1 (EIF2S1) is among these candidates. EIF2S1 is known as a master regulator of cellular stress responses that additionally regulates circadian rhythms. We show that knockdown of EIF2S1 furthermore impairs temperature compensation, suggesting that the role of CPSF6 in temperature compensation may be mediated by its regulation of EIF2S1.

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Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4467-4472.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4467-4472

Author(s):

M Altmann ◽

N Sonenberg ◽

H Trachsel

Keyword(s):

Saccharomyces Cerevisiae ◽

Point Mutations ◽

Translation Initiation Factor ◽

Apparent Temperature ◽

Initiation Factor ◽

Temperature Sensitive ◽

Wild Type ◽

Free System ◽

Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

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Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4525 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4525-4535 ◽

Cited By ~ 23

Author(s):

D R Evans ◽

C Rasmussen ◽

P J Hanic-Joyce ◽

G C Johnston ◽

R A Singer ◽

...

Keyword(s):

Translation Initiation ◽

Mutational Analysis ◽

Dominant Negative ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Temperature Sensitive ◽

Gene Transcript ◽

Missense Mutations ◽

Wild Type ◽

Protein Subunit

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

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Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4467 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4467-4472 ◽

Cited By ~ 61

Author(s):

M Altmann ◽

N Sonenberg ◽

H Trachsel

Keyword(s):

Saccharomyces Cerevisiae ◽

Point Mutations ◽

Translation Initiation Factor ◽

Apparent Temperature ◽

Initiation Factor ◽

Temperature Sensitive ◽

Wild Type ◽

Free System ◽

Gal1 Promoter

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CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

Biochemical Journal ◽

10.1042/bj3400135 ◽

1999 ◽

Vol 340 (1) ◽

pp. 135-141 ◽

Cited By ~ 23

Author(s):

Parisa DANAIE ◽

Michael ALTMANN ◽

Michael N. HALL ◽

Hans TRACHSEL ◽

Stephen B. HELLIWELL

Keyword(s):

Cell Cycle ◽

S Phase ◽

Translation Initiation Factor ◽

Yeast Cells ◽

Initiation Factor ◽

G1 Phase ◽

Mrna Levels ◽

Wild Type ◽

Phase Progression ◽

Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

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Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3463 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3463-3471 ◽

Cited By ~ 62

Author(s):

S R Schmid ◽

P Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Clear Correlation ◽

Temperature Sensitive ◽

Rna Helicases ◽

Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

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Mammalian orthoreovirus Infection is Enhanced in Cells Pre-Treated with Sodium Arsenite

Viruses ◽

10.3390/v11060563 ◽

2019 ◽

Vol 11 (6) ◽

pp. 563 ◽

Cited By ~ 2

Author(s):

Michael M. Lutz ◽

Megan P. Worth ◽

Meleana M. Hinchman ◽

John S.L. Parker ◽

Emily D. Ledgerwood

Keyword(s):

Protein Expression ◽

Stress Responses ◽

Viral Protein ◽

Sodium Arsenite ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Viral Protein Expression ◽

Viral Yield ◽

Pre Treatment ◽

In Cells

Following reovirus infection, cells activate stress responses that repress canonical translation as a mechanism to limit progeny virion production. Work by others suggests that these stress responses, which are part of the integrated stress response (ISR), may benefit rather than repress reovirus replication. Here, we report that compared to untreated cells, treating cells with sodium arsenite (SA) to activate the ISR prior to infection enhanced viral protein expression, percent infectivity, and viral titer. SA-mediated enhancement was not strain-specific, but was cell-type specific. While SA pre-treatment of cells offered the greatest enhancement, treatment within the first 4 h of infection increased the percent of cells infected. SA activates the heme-regulated eIF2α (HRI) kinase, which phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) to induce stress granule (SG) formation. Heat shock (HS), another activator of HRI, also induced eIF2α phosphorylation and SGs in cells. However, HS had no effect on percent infectivity or viral yield but did enhance viral protein expression. These data suggest that SA pre-treatment perturbs the cell in a way that is beneficial for reovirus and that this enhancement is independent of SG induction. Understanding how to manipulate the cellular stress responses during infection to enhance replication could help to maximize the oncolytic potential of reovirus.

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Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2

International Journal of Molecular Sciences ◽

10.3390/ijms21030735 ◽

2020 ◽

Vol 21 (3) ◽

pp. 735

Author(s):

Jerneja Tomsic ◽

Arianna Smorlesi ◽

Enrico Caserta ◽

Anna Maria Giuliodori ◽

Cynthia L. Pon ◽

...

Keyword(s):

Amino Acids ◽

Cell Viability ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Geobacillus Stearothermophilus ◽

Wild Type ◽

Initiation Complex ◽

Structural Alterations

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

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Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein

Journal of General Virology ◽

10.1099/vir.0.041830-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1483-1494 ◽

Cited By ~ 21

Author(s):

Michelle M. Arnold ◽

Catie Small Brownback ◽

Zenobia F. Taraporewala ◽

John T. Patton

Keyword(s):

Binding Protein ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Host Protein ◽

Initiation Factor ◽

Nuclear Accumulation ◽

Progeny Virus ◽

Wild Type ◽

Isolation And Characterization ◽

Infected Cells

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3′-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines.

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Regulation of Antioxidant Metabolism by Translation Initiation Factor 2α

The Journal of Cell Biology ◽

10.1083/jcb.152.5.997 ◽

2001 ◽

Vol 152 (5) ◽

pp. 997-1006 ◽

Cited By ~ 50

Author(s):

Shirlee Tan ◽

Nikunj Somia ◽

Pamela Maher ◽

David Schubert

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Nerve Cell ◽

Translation Initiation ◽

Cell Model ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Expression Cloning ◽

Wild Type ◽

Antisense Gene

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress–induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2α and express a low amount of eIF2α. Sensitivity is restored when the clones are transfected with full-length eIF2α; transfection of wild-type cells with the truncated eIF2α gene confers resistance. The phosphorylation of eIF2α also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca2+. In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca2+ when stressed, and the GSH synthetic enzyme γ-glutamyl cysteine synthetase (γGCS) is elevated. The change in γGCS is regulated by a translational mechanism. Therefore, eIF2α is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

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Melanoma differentiation-associated gene 5 is involved in the induction of stress granules and autophagy by protonophore CCCP

Biological Chemistry ◽

10.1515/hsz-2015-0195 ◽

2016 ◽

Vol 397 (1) ◽

pp. 67-74 ◽

Cited By ~ 1

Author(s):

Feng Xu ◽

Xiaobo Li ◽

Peifen Zhang ◽

Jun Xia ◽

Yi Wang ◽

...

Keyword(s):

Innate Immunity ◽

Stress Responses ◽

Stress Granules ◽

Eukaryotic Cell ◽

Innate Immune ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

External Stimuli

Abstract The eukaryotic cell has evolved a variety of stress responses against external stimuli, such as innate immunity, the formation of stress granules (SGs), and autophagy. We previously demonstrated that the innate immune adaptor IFN-β promoter stimulator 1 (IPS-1) plays an essential role in the formation of dsRNA-induced SGs, indicating a connection between SG formation and innate immunity. In this study, it was further demonstrated that melanoma differentiation-associated gene 5 (MDA5), an innate immune sensor, is involved in SG formation induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore. MDA5 knockdown had no significant impact on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) triggered by CCCP, and MDA5 itself was not recruited to SGs, suggesting that the regulation of MDA5 in the SG response occurs downstream of eIF2α. Furthermore, the depletion of MDA5 or G3BP1 led to reduced autophagy in CCCP-stimulated cells, implying that the regulatory effect of MDA5 with respect to autophagy depends on its role in SG formation. This study uncovered an unexpected role of the innate immune protein MDA5 in SG formation and autophagy triggered by the protonophore CCCP, further supporting a correlation between different stress responses.

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