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 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 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 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 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 Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp

2005 ◽  
Vol 79 (2) ◽  
pp. 677-683 ◽  
Author(s):  
Randal C. Cevallos ◽  
Peter Sarnow
Keyword(s):  
Internal Ribosome Entry Site ◽  
Intergenic Region ◽  
Rna Virus ◽  
Penaeid Shrimp ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Functional Conservation

ABSTRACT The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

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

scholarly journals Identification of Internal Ribosome Entry Segment (IRES)-trans-Acting Factors for the Myc Family of IRESs

2007 ◽  
Vol 28 (1) ◽  
pp. 40-49 ◽  
Author(s):  
Laura C. Cobbold ◽  
Keith A. Spriggs ◽  
Stephen J. Haines ◽  
Helen C. Dobbyn ◽  
Christopher Hayes ◽  
...  
Keyword(s):  
Binding Protein ◽  
Structural Element ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Binding Factor ◽  
Internal Ribosome Entry Segment ◽  
Complex Structural ◽  
Rna Chaperones

ABSTRACT The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.

scholarly journals Rous Sarcoma Virus Translation Revisited: Characterization of an Internal Ribosome Entry Segment in the 5′ Leader of the Genomic RNA

2000 ◽  
Vol 74 (24) ◽  
pp. 11581-11588 ◽  
Author(s):  
Clarence Deffaud ◽  
Jean-Luc Darlix
Keyword(s):  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Secondary Structures ◽  
Open Reading Frames ◽  
Start Codon ◽  
Genomic Rna ◽  
Internal Ribosome Entry ◽  
Rous Sarcoma ◽  
Internal Ribosome Entry Segment ◽  
Sarcoma Virus

ABSTRACT The 5′ leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5′ cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5′ end of the genomic RNA directs efficient Gag expression despite stable 5′ secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5′ leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5′ cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3′ cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5′ and 3′ leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3′ sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.

scholarly journals Transcript-Selective Translational Silencing by Gamma Interferon Is Directed by a Novel Structural Element in the Ceruloplasmin mRNA 3′ Untranslated Region

2003 ◽  
Vol 23 (5) ◽  
pp. 1509-1519 ◽  
Author(s):  
Prabha Sampath ◽  
Barsanjit Mazumder ◽  
Vasudevan Seshadri ◽  
Paul L. Fox
Keyword(s):  
Translational Control ◽  
Untranslated Region ◽  
Gamma Interferon ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Structural Element ◽  
Local Inflammatory Response ◽  
Ifn Γ

ABSTRACT Transcript-selective translational control of eukaryotic gene expression is often directed by a structural element in the 3′ untranslated region (3′-UTR) of the mRNA. In the case of ceruloplasmin (Cp), induced synthesis of the protein by gamma interferon (IFN-γ) in U937 monocytic cells is halted by a delayed translational silencing mechanism requiring the binding of a cytosolic inhibitor to the Cp 3′-UTR. Silencing requires the essential elements of mRNA circularization, i.e., eukaryotic initiation factor 4G, poly(A)-binding protein, and poly(A) tail. We here determined the minimal silencing element in the Cp 3′-UTR by progressive deletions from both termini. A minimal, 29-nucleotide (nt) element was determined by gel shift assay to be sufficient for maximal binding of the IFN-γ-activated inhibitor of translation (GAIT), an as-yet-unidentified protein or complex. The interaction was shown to be functional by an in vitro translation assay in which the GAIT element was used as a decoy to overcome translational silencing. Mutation analysis showed that the GAIT element contained a 5-nt terminal loop, a weak 3-bp helix, an asymmetric internal bulge, and a proximal 6-bp helical stem. Two invariant loop residues essential for binding activity were identified. Ligation of the GAIT element immediately downstream of a luciferase reporter conferred the translational silencing response to the heterologous transcript in vitro and in vivo; a construct containing a nonbinding, mutated GAIT element was ineffective. Translational silencing of Cp, and possibly other transcripts, mediated by the GAIT element may contribute to the resolution of the local inflammatory response following cytokine activation of macrophages.

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