A new internal-ribosome-entry-site motif potentiates XIAP- mediated cytoprotection

Abstract

rogrammed cell death (apoptosis) plays a critical part in regulating cell turnover during embryogenesis, metamorphosis, tissue homeostasis and viral infection 1 . Dysregulation of apoptosis occurs in such pathologies as cancer, autoimmunity, immunodeficiency and neurodegeneration. Proteins of the inhibitor-ofapoptosis (IAP) family are intrinsic cellular suppressors of apoptosis and are represented by highly conserved members found from insect viruses to mammals 2-4 . The most potent mammalian IAP is the X-linked IAP, or XIAP 5 , whose mechanism of action involves direct inhibition of caspases 3 and 7, key proteases of the apoptotic cascade 6 . Cellular control of XIAP expression should be fundamental to a cell's ability to modulate its responses to apoptotic stimuli. However, XIAP messenger RNA is expressed in most tissues and cells at fairly constant levels 5 , indicating that translational control of XIAP levels may be an important regulatory mechanism. Here we characterize the primary genomic structure and function of XIAP, and show that XIAP expression is controlled at the translational level, specifically through an internal ribosome-entry site (IRES).Several features of XIAP mRNA indicate that it may be translationally regulated, including an unusually long 5′ untranslated region (UTR) (>5.5 kilobases (kb) for murine and >1.6 kb for human XIAP transcripts) with predicted complex secondary structure and numerous potential translation start sites upstream of the authentic initiation codon. This UTR would be expected to present a significant obstacle to efficient translation by conventional ribosome scanning 7 . An alternative mechanism of translation initiation, mediated through the IRES, has been identified in picornaviruses and in a few cellular mRNAs 8 . Thus we tested whether the 5′ UTR of XIAP mRNA could be involved in translation initiation from reporter-based bicistronic mRNA transcripts encoding β-galactosidase and chloramphenicol aceytyltransferase (CAT) (for example, see ref. 9). (Translation of β-galactosidase is driven by the 5′ mRNA methylguanosine cap.) Both human and mouse XIAP 5′ UTRs directed translation of the second cistron (encoding CAT) at 150-fold higher levels than those produced without the 5′ UTR or with the 5′ UTR in reverse orientation, suggesting the presence of an IRES (Fig. 1a). No activity was detected when using the identical DNA segments cloned into a promoterless construct, confirming P Figure 1 The 5′ UTR of mouse and human XIAP mRNA contains functional IRES elements. a, DNA segments corresponding to the indicated regions of the 5′ UTR of human (h) or mouse (m) XIAP transcripts were inserted (in the indicated directions) into the XhoI site of the linker region (LR) of the bicistronic plasmid pβgal/CAT (where βgal is β-galactosidase); HeLa cells were transfected with these plasmids (2 µg each). The promoterless CAT reporter plasmid pCATbasic/UTR was constructed by inserting the indicated 5′ UTR region into the pCATbasic vector, and in this case HeLa cells were co-transfected ...

Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation

“…As certain types of cell stress pathways are known to influence mechanisms that control mRNA translation, and as p53 function is reliant on its capacity to rapidly respond to cell stress, it is not surprising that p53 mRNA translation is responsive to cell stress. For example, the expression of apoptosis linked APAF-1 (Coldwell et al, 2000), X-linked inhibitor of apoptosis protein (Holcik et al, 1999) as well as the hypoxia response factor hypoxia-inducible factor-a are regulated through IRESs (Lang et al, 2002). Hence, the capacity of the p53 mRNA to coordinate the synthesis of p53 products with the synthesis of executing stress-response factors could form an important part in the p53 response to changes in the cellular environment.…”

A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: A novel mechanism of oncogene de-regulation

“…The latter is mediated by structured regions of RNA termed internal ribosome entry segments (IRES), and these have the ability to direct ribosome binding some considerable distance (up to 1000 nts) from the 5' end of the message. IRESs were originally discovered in picornaviral mRNAs, but have now been identi®ed in a number of eukaryotic mRNAs, the protein products of which are involved in the control of cell growth including, FGF-2 (Vagner et al, 1995), VEGF (Miller et al, 1998;Stein et al, 1998), PDGF (Bernstein et al, 1997) and c-myc (Nanbru et al, 1997;Stoneley et al, 1998) and cell death Holcik et al, 1999). In the case of c-myc we have shown that initiation of protein synthesis can occur by two distinct mechanisms; cap-dependent scanning and IRES mediated .…”

Reversible induction of translational isoforms of p53 in glucose deprivation

“…29 Serum starvation of mammalian cell cultures showed induction of Bcl-2 IRES 30 and activated translation of XIAP mRNA. 31 IRES-mediated translation of p27 Kip1 mRNA contributes to maintenance of G1 phase of the cell cycle and the expression of p27 Kip1 was found to be iron sensitive. 32,33 These studies reveal a novel aspect of activation of IRESmediated translation of eukaryotic mRNAs due to nutrient shortage, resulting in the synthesis of proteins essential for cell survival or apoptosis.…”