Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5′- untranslated regions (5′-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5′-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5′-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.

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Ribosomal RACK1:Protein Kinase C βII Modulates Intramolecular Interactions between Unstructured Regions of Eukaryotic Initiation Factor 4G (eIF4G) That Control eIF4E and eIF3 Binding

Molecular and Cellular Biology ◽

10.1128/mcb.00306-18 ◽

2018 ◽

Vol 38 (19) ◽

Cited By ~ 11

Author(s):

Mikhail I. Dobrikov ◽

Elena Y. Dobrikova ◽

Matthias Gromeier

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Intramolecular Interactions ◽

Initiation Factor ◽

Binding Motif ◽

Eukaryotic Initiation Factor ◽

Kinase C ◽

Eukaryotic Initiation Factor 4G ◽

Β Sheet ◽

Unstructured Regions

ABSTRACT The receptor for activated C kinase (RACK1), a conserved constituent of eukaryotic ribosomes, mediates phosphorylation of eukaryotic initiation factor 4G1(S1093) [eIF4G1(S1093)] and eIF3a(S1364) by protein kinase C βII (PKCβII) (M. I. Dobrikov, E. Y. Dobrikova, and M. Gromeier, Mol Cell Biol 38:e00304-18, 2018, https://doi.org/10.1128/MCB.00304-18). RACK1:PKCβII activation drives a phorbol ester-induced surge of global protein synthesis and template-specific translation induction of PKC–Raf–extracellular signal-regulated kinase 1/2 (ERK1/2)-responsive genes. For unraveling mechanisms of RACK1:PKCβII-mediated translation stimulation, we used sequentially truncated eIF4G1 in coimmunoprecipitation analyses to delineate a set of autoinhibitory elements in the N-terminal unstructured region (surrounding the eIF4E-binding motif) and the interdomain linker (within the eIF3-binding site) of eIF4G1. Computer-based predictions of secondary structure, mutational analyses, and fluorescent titration with the β-sheet dye thioflavin T suggest that eIF4G1(S1093) modulates a 4-stranded β-sheet composed of antiparallel β-hairpins formed by the autoinhibitory elements in eIF4G1's unstructured regions. The intact β-sheet “locks” the eIF4G configuration, preventing assembly with eIF3/40S ribosomal subunits. Upon PKC stimulation, activated RACK1:PKCβII phosphorylates eIF4G(S1093) in the tight 48S initiation complex, possibly facilitating dissociation/recycling of eIF4F.

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

Biochemical Journal ◽

10.1042/bj20040769 ◽

2005 ◽

Vol 385 (3) ◽

pp. 695-702 ◽

Cited By ~ 9

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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RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

Nucleic Acids Research ◽

10.1093/nar/gkaa646 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8562-8575 ◽

Cited By ~ 2

Author(s):

Francis Robert ◽

Regina Cencic ◽

Renying Cai ◽

T Martin Schmeing ◽

Jerry Pelletier

Keyword(s):

Translation Initiation ◽

Mammalian Cells ◽

Structural Information ◽

Critical Role ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Eif4f Complex ◽

Cellular Mrnas ◽

Open Question ◽

Reporter Mrna

Abstract Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.

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