Mapping and characterization of the minimal internal ribosome entry segment in the human c-myc mRNA 5′ untranslated region

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.

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

Oncogene ◽

10.1038/sj.onc.1204157 ◽

2001 ◽

Vol 20 (7) ◽

pp. 893-897 ◽

Cited By ~ 3

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

Molecular and Cellular Biology ◽

10.1128/mcb.01283-08 ◽

2009 ◽

Vol 29 (6) ◽

pp. 1565-1574 ◽

Cited By ~ 42

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1162-1169.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1162-1169 ◽

Cited By ~ 135

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.

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Mutator-Suppressible Alleles of rough sheath1 and liguleless3 in Maize Reveal Multiple Mechanisms for Suppression

Genetics ◽

10.1093/genetics/154.1.437 ◽

2000 ◽

Vol 154 (1) ◽

pp. 437-446 ◽

Cited By ~ 1

Author(s):

Lisa Girard ◽

Michael Freeling

Keyword(s):

Untranslated Region ◽

Mutant Allele ◽

Negative Regulation ◽

Wild Type ◽

Knox Gene ◽

Transcript Accumulation ◽

Mu Suppression ◽

Different Types ◽

Rna Blot Analysis

Abstract Insertions of Mutator transposons into maize genes can generate suppressible alleles. Mu suppression is when, in the absence of Mu activity, the phenotype of a mutant allele reverts to that of its progenitor. Here we present the characterization of five dominant Mu-suppressible alleles of the knox (knotted1-like homeobox) genes liguleless3 and rough sheath1, which exhibit neomorphic phenotypes in the leaves. RNA blot analysis suggests that Mu suppression affects only the neomorphic aspect of the allele, not the wild-type aspect. Additionally, Mu suppression appears to be exerting its effects at the level of transcription or transcript accumulation. We show that truncated transcripts are produced by three alleles, implying a mechanism for Mu suppression of 5′ untranslated region insertion alleles distinct from that which has been described previously. Additionally, it is found that Mu suppression can be caused by at least three different types of Mutator elements. Evidence presented here suggests that whether an allele is suppressible or not may depend upon the site of insertion. We cite previous work on the knox gene kn1, and discuss our results in the context of interactions between Mu-encoded products and the inherently negative regulation of neomorphic liguleless3 and rough sheath1 transcription.

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Poliovirus Internal Ribosome Entry Segment Structure Alterations That Specifically Affect Function in Neuronal Cells: Molecular Genetic Analysis

Journal of Virology ◽

10.1128/jvi.76.21.10617-10626.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10617-10626 ◽

Cited By ~ 37

Author(s):

Cécile E. Malnou ◽

Tuija A. A. Pöyry ◽

Richard J. Jackson ◽

Katherine M. Kean

Keyword(s):

Secondary Structure ◽

Neuronal Cells ◽

Neuronal Cell ◽

In Vitro Translation ◽

Internal Ribosome Entry ◽

Cell Extracts ◽

Internal Ribosome Entry Segment ◽

Domain V ◽

Bulge Loop

ABSTRACT Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5′ noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5°C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.

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