scholarly journals The 5′ Untranslated Region of Protein Kinase Cδ Directs Translation by an Internal Ribosome Entry Segment That Is Most Active in Densely Growing Cells and during Apoptosis

2002 ◽  
Vol 22 (17) ◽  
pp. 6089-6099 ◽  
Author(s):  
Bronwyn C. Morrish ◽  
Martin G. Rumsby
Keyword(s):  
Protein Kinase ◽  
Untranslated Region ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Serum Starvation ◽  
Proliferating Cells ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Protein Kinase Cδ

ABSTRACT Protein kinase Cδ (PKCδ) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5′ untranslated region (5′ UTR) of PKCδ is predicted to form very stable secondary structures with free energies (ΔG values) of around −170 kcal/mol. The 5′ UTR of PKCδ can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5′ UTR of PKCδ contains a functional internal ribosome entry segment (IRES). The activity of the PKCδ IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5′ UTR of PKCδ is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCδ has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCδ synthesis can be maintained under these conditions when cap-dependent translation is inhibited.

scholarly journals Canonical Initiation Factor Requirements of the Myc Family of Internal Ribosome Entry Segments

2009 ◽  
Vol 29 (6) ◽  
pp. 1565-1574 ◽  
Author(s):  
Keith A. Spriggs ◽  
Laura C. Cobbold ◽  
Catherine L. Jopling ◽  
Rebecca E. Cooper ◽  
Lindsay A. Wilson ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Ternary Complex ◽  
Initiation Factor ◽  
Start Codon ◽  
Structural Element ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Eif4f Complex ◽  
Internal Ribosome Entry Segment ◽  
Minimum Requirements

ABSTRACT Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5′ end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5′ untranslated region (5′-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5′-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.

scholarly journals c-Myc Protein Synthesis Is Initiated from the Internal Ribosome Entry Segment during Apoptosis

2000 ◽  
Vol 20 (4) ◽  
pp. 1162-1169 ◽  
Author(s):  
Mark Stoneley ◽  
Stephen A. Chappell ◽  
Catherine L. Jopling ◽  
Martin Dickens ◽  
Marion MacFarlane ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
P38 Mapk ◽  
Untranslated Region ◽  
Mapk Pathway ◽  
Specific Inhibitor ◽  
Initiation Factor ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation ◽  
Apoptosis Protein ◽  
Internal Ribosome Entry Segment

ABSTRACT Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5′ untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5′ untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.

scholarly journals Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation

2001 ◽  
Vol 21 (13) ◽  
pp. 4097-4109 ◽  
Author(s):  
Yanne M. Michel ◽  
Andrew M. Borman ◽  
Sylvie Paulous ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Initiation Factor ◽  
Translation System ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Hcv Ires ◽  
Stimulation Of

ABSTRACT Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5′ m7GpppN cap and the 3′ poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3′ end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3′-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

scholarly journals C-Myc 5′ untranslated region contains an internal ribosome entry segment

Oncogene ◽  
1998 ◽  
Vol 16 (3) ◽  
pp. 423-428 ◽  
Author(s):  
Mark Stoneley ◽  
Fiona EM Paulin ◽  
John PC Le Quesne ◽  
Stephen A Chappell ◽  
Anne E Willis
Keyword(s):  
Untranslated Region ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment

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

2018 ◽  
Vol 38 (19) ◽  
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.

scholarly journals Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5′-untranslated region of mRNA

2004 ◽  
Vol 378 (1) ◽  
pp. 73-82 ◽  
Author(s):  
William J. TUXWORTH ◽  
Atif N. SAGHIR ◽  
Laura S. SPRUILL ◽  
Donald R. MENICK ◽  
Paul J. McDERMOTT
Keyword(s):  
Protein Synthesis ◽  
Secondary Structure ◽  
Untranslated Region ◽  
Primary Cultures ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Hypertrophic Growth ◽  
Reporter Mrna

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5´-UTR (5´-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either ‘stronger’ (less structure in the 5´-UTR) or ‘weaker’ (more structure in the 5´-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5´-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48±13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5´-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

scholarly journals Detailed Analysis of the Requirements of Hepatitis A Virus Internal Ribosome Entry Segment for the Eukaryotic Initiation Factor Complex eIF4F

2001 ◽  
Vol 75 (17) ◽  
pp. 7864-7871 ◽  
Author(s):  
Andrew M. Borman ◽  
Yanne M. Michel ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Eif4f Complex ◽  
Internal Ribosome Entry Segment

ABSTRACT The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

scholarly journals Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent

2001 ◽  
Vol 75 (24) ◽  
pp. 12141-12152 ◽  
Author(s):  
Daniel R. Gallie
Keyword(s):  
Large Subunit ◽  
Tobacco Etch Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4G ◽  
Independent Translation

ABSTRACT The 5′ leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

scholarly journals Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

2014 ◽  
Vol 52 (3) ◽  
pp. 373-382 ◽  
Author(s):  
Kelly León ◽  
Thomas Boulo ◽  
Astrid Musnier ◽  
Julia Morales ◽  
Christophe Gauthier ◽  
...  
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Translational Machinery ◽  
Phosphorylation Event ◽  
Eukaryotic Initiation Factor 4G ◽  
Molecular Bases

The control of mRNA translation has been mainly explored in response to activated tyrosine kinase receptors. In contrast, mechanistic details on the translational machinery are far less available in the case of ligand-bound G protein-coupled receptors (GPCRs). In this study, using the FSH receptor (FSH-R) as a model receptor, we demonstrate that part of the translational regulations occurs by phosphorylation of the translation pre-initiation complex scaffold protein, eukaryotic initiation factor 4G (eIF4G), in HEK293 cells stably expressing the FSH-R. This phosphorylation event occurred when eIF4G was bound to the mRNA 5′ cap, and probably involves mammalian target of rapamycin. This regulation might contribute to cap-dependent translation in response to FSH. The cap-binding protein eIF4E also had its phosphorylation level enhanced upon FSH stimulation. We also show that FSH-induced signaling not only led to cap-dependent translation but also to internal ribosome entry site (IRES)-dependent translation of some mRNA. These data add detailed information on the molecular bases underlying the regulation of selective mRNA translation by a GPCR, and a topological model recapitulating these mechanisms is proposed.

scholarly journals An internal ribosome entry segment in the 5′ untranslated region of the mnt gene

Oncogene ◽  
2001 ◽  
Vol 20 (7) ◽  
pp. 893-897 ◽  
Author(s):  
Mark Stoneley ◽  
Jonathan P Spencer ◽  
Stephanie C Wright
Keyword(s):  
Untranslated Region ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment
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