Translation of the human c-myc P0 tricistronic mRNA involves two independent internal ribosome entry sites.

The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.     

A high-efficiency translational control element with potential for cancer gene therapy

An active internal ribosome entry sequence (IRES) that efficiently mediates cap (m7pppGN)-independent translation in human carcinoma cells could be an effective device for gene co-transduction in cancer gene therapy. In this study using the cytomegalovirus (CMV) promoter, a remarkable internal translation activity was observed and mediated by the sequence localized to the 183-653 region of 5' NF-kappaB repressor mRNA (NFR183IRES). To test the potential of such sequence for therapeutic application, we carried out in vitro functional assays using the dicistronic constructs that internally expressed human PTEN tumor suppressor. The PTEN expression mediated by NFR183IRES was found to result in growth inhibition of carcinoma cells more effectively than the expression by NFR1IRES that contained the 1-653 region. When compared to the internal translation driven by the picornaviral IRES element of the encephalomyocarditis virus (EMCV) or the foot-and-mouth disease virus (FMDV), NFR183IRES consistently exhibited a higher activity in the human carcinoma cells, HeLa, LNCaP and MCF7. Such high-efficiency translational control element may prove useful for cancer gene therapy.     

Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon

The expression of the cyclin-dependent kinase inhibitor p15INK4B in normal cells after induction with TGF-beta1, or following overexpression from an adenovirus-encoded cDNA, appears on an SDS-polyacrylamide gel as a doublet. Here, the underlying mechanism behind the synthesis of the two species has been studied. By expressing cDNAs truncated at their 5' end, we found that the synthesis of the more slowly migrating form, called p15.5INK4B, is dependent on a sequence upstream of the first AUG codon thought to initiate translation of p15INK4B. Two potential, in frame, alternative upstream initiation codons, ACG and GUG, were individually changed to GCA encoding alanine. Analysis by in vitro translation, or immunoblotting of lysates from transfected 293 cells, showed that translation of p15.5INK4B is initiated at the GUG located 13 codons upstream of the first AUG. When this AUG was mutated, p15INK4B was no longer made. Instead, a shorter form, initiated at an in frame AUG located seven codons downstream, was synthesized. Finally, when both these AUGs were mutated, only p15.5INK4B was generated. Both p15INK4B and p15.5INK4B bound to CDK4 and CDK6, inhibited DNA synthesis, and caused replicative senescence of a human glioma cell line. We thus conclude that p15INK4B and p15.5INK4B, encoded by the CDKN2B gene, are functionally indistinguishable as based on these assays.     

Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5' untranslated region (5'UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5'UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)-mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G(1). When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity.     

Initiation of Apaf-1 translation by internal ribosome entry

The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5' untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5' UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3' end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised. Oncogene (2000) 19, 899 - 905.