scholarly journals Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea

2022 ◽  
Vol 8 ◽  
Author(s):  
Xiaozhuo Liu ◽  
Houtan Moshiri ◽  
Qian He ◽  
Ansuman Sahoo ◽  
Sarah E. Walker
Keyword(s):  
Gene Ontology ◽  
Stress Responses ◽  
Mammalian Cells ◽  
Growth Conditions ◽  
Initiation Factor ◽  
Specific Gene ◽  
Rna Recognition Motif ◽  
Eukaryotic Initiation Factor ◽  
Terminal Domain ◽  
Mrna Pool

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA recruitment. Recent evidence indicates yeast mRNAs have variable dependence on eIF4B under optimal growth conditions. Given the ability of eIF4B to promote translation as a function of nutrient conditions in mammalian cells, we wondered if eIF4B activities in translation could alter phenotypes in yeast through differential mRNA selection for translation. Here we compared the effects of disrupting yeast eIF4B RNA- and 40S-binding motifs under ∼1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to stressors requiring robust cellular integrity. In particular, the NTD conferred a strong growth advantage in the presence of urea, which may be important for pathogenesis of related fungal species. Ribosome profiling indicated that similar to complete eIF4B deletion, deletion of the NTD dramatically reduced translation, particularly of those mRNAs with long and highly structured 5-prime untranslated regions. This behavior was observed both with and without urea exposure, but the specific mRNA pool associated with ribosomes in response to urea differed. Deletion of the NTD led to relative increases in ribosome association of shorter transcripts with higher dependence on eIF4G, as was noted previously for eIF4B deletion. Gene ontology analysis indicated that proteins encoded by eIF4B NTD-dependent transcripts were associated with the cellular membrane system and the cell wall, while NTD-independent transcripts encoded proteins associated with cytoplasmic proteins and protein synthesis. This analysis highlighted the difference in structure content of mRNAs encoding membrane versus cytoplasmic housekeeping proteins and the variable reliance of specific gene ontology classes on various initiation factors promoting otherwise similar functions. Together our analyses suggest that deletion of the eIF4B NTD prevents cellular stress responses by affecting the capacity to translate a diverse mRNA pool.

scholarly journals Binding to the Ribosome by Eukaryotic Initiation Factor 4B Drives Yeast Translational Control in Response to Urea

2019 ◽  
Author(s):  
Xiaozhuo Liu ◽  
Houtan Moshiri ◽  
Sarah Walker
Keyword(s):  
Stress Responses ◽  
Translational Control ◽  
Growth Conditions ◽  
Ribosome Profiling ◽  
Initiation Factor ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Eukaryotic Initiation Factor ◽  
Ribosome Binding ◽  
Mrna Binding

ABSTRACTThe yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA binding. Recent evidence suggests mRNAs have variable dependence on eIF4B, suggesting this factor could promote changes in mRNA selection to adapt to stressors. However, the importance of eIF4B and its constituent domains for mRNA selection under diverse cellular and environmental conditions remain undefined. Here we compared the effects of disrupting eIF4B RNA- and ribosome-binding under ∼1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but ribosome binding by the N-terminal Domain (NTD) promoted growth in response to various stressors. In particular, the NTD conferred a strong growth advantage in the presence of urea. Ribosome profiling revealed that the NTD promoted translation of mRNAs with long and highly structured 5-prime untranslated regions, both with and without urea exposure. Because these changes required 40S binding, our results suggest eIF4B regulates mRNA loading and scanning as a part of the PIC, rather than by activating mRNPs prior to ribosome binding. Furthermore, our data indicate the yeast response to urea includes a translational component, driven by translation of mRNAs encoding proteins associated with the cellular periphery, suggesting general eIFs can promote diverse cellular responses.

scholarly journals The C-Terminal Region of Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes mRNA Recruitment, Scanning, and, Together with eIF3j and the eIF3b RNA Recognition Motif, Selection of AUG Start Codons

2010 ◽  
Vol 30 (18) ◽  
pp. 4415-4434 ◽  
Author(s):  
Wen-Ling Chiu ◽  
Susan Wagner ◽  
Anna Herrmannová ◽  
Laxminarayana Burela ◽  
Fan Zhang ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Recognition Motif ◽  
Terminal Domain ◽  
Eukaryotic Translation Initiation

ABSTRACT The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.

scholarly journals Cloning and expression of cDNAs for the β subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2

1995 ◽  
Vol 309 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
B L Craddock ◽  
N T Price ◽  
C G Proud
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes. In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta. Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested.

scholarly journals Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain

2021 ◽  
Vol 12 ◽  
Author(s):  
Nathan M. Doner ◽  
Damien Seay ◽  
Marina Mehling ◽  
Siqi Sun ◽  
Satinder K. Gidda ◽  
...  
Keyword(s):  
Stress Responses ◽  
Mammalian Cells ◽  
Lipid Droplets ◽  
Plant Stress ◽  
Plant Cells ◽  
Normal Growth ◽  
Cell Types ◽  
Growth Conditions ◽  
Functional Aspects ◽  
Necessary And Sufficient

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

scholarly journals Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)

2013 ◽  
Vol 289 (3) ◽  
pp. 1704-1722 ◽  
Author(s):  
Fujun Zhou ◽  
Sarah E. Walker ◽  
Sarah F. Mitchell ◽  
Jon R. Lorsch ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Rna Recognition Motif ◽  
Conserved Motifs ◽  
Terminal Domain ◽  
Sequence Elements

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

scholarly journals Selective Modification of Eukaryotic Initiation Factor 4F (eIF4F) at the Onset of Cell Differentiation: Recruitment of eIF4GII and Long-Lasting Phosphorylation of eIF4E

2004 ◽  
Vol 24 (11) ◽  
pp. 4920-4928 ◽  
Author(s):  
Sandrine Caron ◽  
Martine Charon ◽  
Elisabeth Cramer ◽  
Nahum Sonenberg ◽  
Isabelle Dusanter-Fourt
Keyword(s):  
Functional Analysis ◽  
Cell Differentiation ◽  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Scaffold Protein ◽  
Synergistic Action ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Critical Link

ABSTRACT mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5′ cap structure and the 3′ poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.

scholarly journals Pancreatic eukaryotic initiation factor-2α kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress

2000 ◽  
Vol 346 (2) ◽  
pp. 281-293 ◽  
Author(s):  
Ruchira SOOD ◽  
Amy C. PORTER ◽  
Kun MA ◽  
Lawrence A. QUILLIAM ◽  
Ronald C. WEK
Keyword(s):  
Endoplasmic Reticulum ◽  
Drosophila Melanogaster ◽  
Caenorhabditis Elegans ◽  
Er Stress ◽  
Translational Control ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Α Subunit

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF-2α). Recently, we identified a new family member, pancreatic eIF-2α kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2α phosphorylation was coincident with increased PEK autophosphorylation and eIF-2α kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2α and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2α kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.

scholarly journals The C-Terminal Domain of Eukaryotic Initiation Factor 5 Promotes Start Codon Recognition by Its Dynamic Interplay with eIF1 and eIF2β

Cell Reports ◽  
2012 ◽  
Vol 1 (6) ◽  
pp. 689-702 ◽  
Author(s):  
Rafael E. Luna ◽  
Haribabu Arthanari ◽  
Hiroyuki Hiraishi ◽  
Jagpreet Nanda ◽  
Pilar Martin-Marcos ◽  
...  
Keyword(s):  
Initiation Factor ◽  
Start Codon ◽  
Eukaryotic Initiation Factor ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Dynamic Interplay

Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

2013 ◽  
Vol 451 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Janete Chung ◽  
Antonio A. Rocha ◽  
Renata R. Tonelli ◽  
Beatriz A. Castilho ◽  
Sergio Schenkman
Keyword(s):  
Protein Synthesis ◽  
Stationary Phase ◽  
Phosphorylation Site ◽  
Elongation Factor ◽  
Growth Conditions ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Wild Type ◽  
Translational Arrest ◽  
Aspartate Residue

The protein known as eIF5A (eukaryotic initiation factor 5A) has an elusive role in translation. It has a unique and essential hypusine modification at a conserved lysine residue in most eukaryotes. In addition, this protein is modified by phosphorylation with unknown functions. In the present study we show that a phosphorylated state of eIF5A predominates in exponentially growing Trypanosoma cruzi cells, and extensive dephosphorylation occurs in cells in stationary phase. Phosphorylation occurs mainly at Ser2, as shown in yeast eIF5A. In addition, a novel phosphorylation site was identified at Tyr21. In exponential cells, T. cruzi eIF5A is partially associated with polysomes, compatible with a proposed function as an elongation factor, and becomes relatively enriched in polysomal fractions in stationary phase. Overexpression of the wild-type eIF5A, or eIF5A with Ser2 replaced by an aspartate residue, but not by alanine, increases the rate of cell proliferation and protein synthesis. However, the presence of an aspartate residue instead of Ser2 is toxic for cells reaching the stationary phase, which show a less-pronounced protein synthesis arrest and a decreased amount of eIF5A in dense fractions of sucrose gradients. We conclude that eIF5A phosphorylation and dephosphorylation cycles regulate translation according to the growth conditions.

scholarly journals The Drosophila protein kinase LK6 is regulated by ERK and phosphorylates the eukaryotic initiation factor eIF4E in vivo

2005 ◽  
Vol 385 (3) ◽  
pp. 695-702 ◽  
Author(s):  
Josep L. PARRA-PALAU ◽  
Gert C. SCHEPER ◽  
Daniel E. HARPER ◽  
Christopher G. PROUD
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Mammalian Cells ◽  
Signalling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Binding Motif ◽  
Eukaryotic Initiation Factor ◽  
Drosophila Cells

In Drosophila cells, phosphorylation of eIF4E (eukaryotic initiation factor 4E) is required for growth and development. In Drosophila melanogaster, LK6 is the closest homologue of mammalian Mnk1 and Mnk2 [MAPK (mitogen-activated protein kinase) signal-integrating kinases 1 and 2 respectively] that phosphorylate mammalian eIF4E. Mnk1 is activated by both mitogen- and stress-activated signalling pathways [ERK (extracellular-signal-regulated kinase) and p38 MAPK], whereas Mnk2 contains a MAPK-binding motif that is selective for ERKs. LK6 possesses a binding motif similar to that in Mnk2. In the present study, we show that LK6 can phosphorylate eIF4E at the physiological site. LK6 activity is increased by the ERK signalling pathway and not by the stress-activated p38 MAPK signalling pathway. Consistent with this, LK6 binds ERK in mammalian cells, and this requires an intact binding motif. LK6 can bind to eIF4G in mammalian cells, and expression of LK6 increases the phosphorylation of the endogenous eIF4E. In Drosophila S2 Schneider cells, LK6 binds the ERK homologue Rolled, but not the p38 MAPK homologue. LK6 phosphorylates Drosophila eIF4E in vitro. The phosphorylation of endogenous eIF4E in Drosophila cells is increased by activation of the ERK pathway but not by arsenite, an activator of p38 MAPK. RNA interference directed against LK6 significantly decreases eIF4E phosphorylation in Drosophila cells. These results show that LK6 binds to ERK and is activated by ERK signalling and it is responsible for phosphorylating eIF4E in Drosophila.

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