scholarly journals PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Teresa Cesaro ◽  
Yohei Hayashi ◽  
Fabian Borghese ◽  
Didier Vertommen ◽  
Fanny Wavreil ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Viral Infections ◽  
Phosphorylation Site ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Poly I:C ◽  
Binding Motif ◽  
Double Stranded Rna

AbstractEukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes.

scholarly journals Specific Anchoring and Local Translation of Poxviral ATI mRNA at Cytoplasmic Inclusion Bodies

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
George C. Katsafanas ◽  
Bernard Moss
Keyword(s):  
Inclusion Bodies ◽  
Actin Polymerization ◽  
Rna Binding ◽  
Protein Localization ◽  
Cytoplasmic Inclusion ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Local Translation ◽  
Virion Assembly

ABSTRACT On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membraneless sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation, and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membraneless protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, relocalization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N terminus of the ATI or large truncations are made at the N or C termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation. IMPORTANCE Poxvirus genome replication, transcription, translation, and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.

scholarly journals Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7601-7612 ◽  
Author(s):  
Sylvie German-Retana ◽  
Jocelyne Walter ◽  
Bénédicte Doublet ◽  
Geneviève Roudet-Tavert ◽  
Valérie Nicaise ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Point Mutations ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Natural Resistance ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

scholarly journals Cancer-associated mRNAs regulated by the Helix-Loop-Helix motif of human EIF3A

2018 ◽  
Author(s):  
Marina Volegova ◽  
Jamie H.D. Cate
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Binding Motif ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Helix Loop Helix ◽  
Small Set

AbstractImproper regulation of translation initiation, a vital check-point of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) has been associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit in eIF3 interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiationin vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, includingMYC. We also demonstrate that the HLH motif of EIF3A acts specifically on the 5’-UTR ofMYCmRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.SummaryThe Helix Loop Helix motif of EIF3A controls translation of a small set of oncogenic cellular transcripts, includingMYC, and modulates the function of translation initiation factor EIF4A1 during translation initiation on select mRNAs.

scholarly journals PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

1997 ◽  
Vol 17 (4) ◽  
pp. 2257-2265 ◽  
Author(s):  
Z J Lorković ◽  
R G Herrmann ◽  
R Oelmüller
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Protein Family ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Helicases ◽  
Nuclear Targeting ◽  
Dead Box ◽  
Cell Extracts ◽  
The Dead

The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

scholarly journals Translational regulation of Chk1 expression by eIF3a via interaction with the RNA-binding protein HuR

2020 ◽  
Vol 477 (10) ◽  
pp. 1939-1950 ◽  
Author(s):  
Zizheng Dong ◽  
Jianguo Liu ◽  
Jian-Ting Zhang
Keyword(s):  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Function ◽  
Rna Recognition Motif ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

eIF3a is a putative subunit of the eukaryotic translation initiation factor 3 complex. Accumulating evidence suggests that eIF3a may have a translational regulatory function by suppressing translation of a subset of mRNAs while accelerating that of other mRNAs. Albeit the suppression of mRNA translation may derive from eIF3a binding to the 5′-UTRs of target mRNAs, how eIF3a may accelerate mRNA translation remains unknown. In this study, we show that eIF3a up-regulates translation of Chk1 but not Chk2 mRNA by interacting with HuR, which binds directly to the 3′-UTR of Chk1 mRNA. The interaction between eIF3a and HuR occurs at the 10-amino-acid repeat domain of eIF3a and the RNA recognition motif domain of HuR. This interaction may effectively circularize Chk1 mRNA to form an end-to-end complex that has recently been suggested to accelerate mRNA translation. Together with previous findings, we conclude that eIF3a may regulate mRNA translation by directly binding to the 5′-UTR to suppress or by interacting with RNA-binding proteins at 3′-UTRs to accelerate mRNA translation.

scholarly journals LncRNA H19 governs mitophagy and restores mitochondrial respiration in the heart through Pink1/Parkin signaling during obesity

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Shao-Hua Wang ◽  
Xiao-Lin Zhu ◽  
Fei Wang ◽  
Si-Xu Chen ◽  
Zhi-Teng Chen ◽  
...  
Keyword(s):  
Respiratory Function ◽  
Metabolic Disorders ◽  
Rna Binding ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Respiratory Capacity ◽  
Mitochondrial Number ◽  
Related Proteins ◽  
Mitochondrial Respiratory

AbstractMaintaining proper mitochondrial respiratory function is crucial for alleviating cardiac metabolic disorders during obesity, and mitophagy is critically involved in this process. Long non-coding RNA H19 (H19) is crucial for metabolic regulation, but its roles in cardiac disorders, mitochondrial respiratory function, and mitophagy during obesity are largely unknown. In this study, palmitic acid (PA)-treated H9c2 cell and Lep−/− mice were used to investigate cardiac metabolic disorders in vitro and in vivo, respectively. The effects of H19 on metabolic disorders, mitochondrial respiratory function, and mitophagy were investigated. Moreover, the regulatory mechanisms of PA, H19, mitophagy, and respiratory function were examined. The models tested displayed a reduction in H19 expression, respiratory function and mitochondrial number and volume, while the expression of mitophagy- and Pink1/Parkin signaling-related proteins was upregulated, as indicated using quantitative real-time PCR, Seahorse mitochondrial stress test analyzer, transmission electron microscopy, fluorescence indicators and western blotting. Forced expression of H19 helped to the recoveries of respiratory capacity and mitochondrial number while inhibited the levels of mitophagy- and Pink1/Parkin signaling-related proteins. Pink1 knockdown also attenuated PA-induced mitophagy and increased respiratory capacity. Mechanistically, RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation assays showed that H19 could hinder the binding of eukaryotic translation initiation factor 4A, isoform 2 (eIF4A2) with Pink1 mRNA, thus inhibiting the translation of Pink1 and attenuation of mitophagy. PA significantly increased the methylation levels of the H19 promoter region by upregulation Dnmt3b methylase levels, thereby inhibiting H19 transcription. Collectively, these findings suggest that DNA methylation-mediated the downregulation of H19 expression plays a crucial role in cardiomyocyte or H9c2 cells metabolic disorders and induces cardiac respiratory dysfunction by promoting mitophagy. H19 inhibits excessive mitophagy by limiting Pink1 mRNA translation, thus alleviating this cardiac defect that occurs during obesity.

scholarly journals Control of the eIF4E activity: structural insights and pharmacological implications

2021 ◽  
Author(s):  
Alice Romagnoli ◽  
Mattia D’Agostino ◽  
Chiara Ardiccioni ◽  
Cristina Maracci ◽  
Stefano Motta ◽  
...  
Keyword(s):  
Fragile X ◽  
Mrna Translation ◽  
Initial Step ◽  
Structural Features ◽  
Autism Spectrum ◽  
Translation Initiation Factor ◽  
Cellular Growth ◽  
Initiation Factor ◽  
Binding Motif ◽  
Translational Machinery

AbstractThe central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E–eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

scholarly journals Murine Cytomegalovirus m142 and m143 Are both Required To Block Protein Kinase R-Mediated Shutdown of Protein Synthesis

2006 ◽  
Vol 80 (20) ◽  
pp. 10181-10190 ◽  
Author(s):  
Ralitsa S. Valchanova ◽  
Marcus Picard-Maureau ◽  
Matthias Budt ◽  
Wolfram Brune
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Rna Binding ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Murine Cytomegalovirus ◽  
Deletion Mutants ◽  
Sequence Motifs ◽  
Protein Kinase R ◽  
Double Stranded Rna

ABSTRACT Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2α, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.

Acute adaptive responses of central sensorimotor neurons after spinal cord injury

2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Kevin Thompson ◽  
Victoria DiBona ◽  
Aditi Dubey ◽  
David Crockett ◽  
Mladen-Roko Rasin
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Rna Binding ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Projection Neurons ◽  
Postnatal Life ◽  
Mrna Levels ◽  
Cord Injury

AbstractSpinal cord injury (SCI) can be a lifelong, devastating condition for both the patient and the caregiver, with a daunting incidence rate. Still, there are only limited available therapies and the effectiveness of precise regeneration within the central nervous system is minimal throughout postnatal life. Recently, improved regeneration after SCI was seen by manipulating a pathway in sensorimotor neocortices that is involved in phosphorylation of an RNA binding protein (RBP) required for mRNA translation, the Eukaryotic translation initiation factor 4E (eIF4E). Our data identifies rapid molecular alterations of eIF4E in the sensorimotor neocortices 1 and 3 days after a lateral hemisection SCI, used as a model for Brown-Séquard syndrome. The function of an RBP depends on both its distribution sites within the cell and its phosphorylation states. Indeed, we found both to be affected after SCI. There was a distinct subcellular redistribution of eIF4E and phosphorylated-eIF4E was reduced, indicating that the eIF4E’s translation was disrupted. Upon identification and analysis of the mRNA cargo of eIF4E in uninjured sensorimotor neocortices, we found that eIF4E binds both Importin-13 (Ipo13) and Parvalbumin (Pv) mRNAs, indicating a role in their translation. Remarkably, eIF4E’s interaction with both Ipo13 and Pv mRNAs was disrupted 1 and 3 days after SCI, despite preservation of total Ipo13 and Pv mRNA levels. Finally, we detected a selective loss of expression of both IPO13 and PV proteins in projection neurons of sensorimotor neocortices, as well as their disrupted dendritic polarity. Since IPO13 is predominantly expressed in neocortical projection neurons and PV in a subset of neocortical interneurons, these data suggest a strong acute effect of SCI on neocortical microcircuitry. Taken together, these data indicate that neocortical eIF4E and a subset of mRNAs may be rapidly recruited to translational machinery after SCI to promote adaptive regeneration response of sensorimotor neurons.

Sign in / Sign up

Export Citation Format

Share Document