A ribosome-related signature in peripheral blood CLL B cells is linked to reduced survival following treatment.

We have used polysome profiling coupled to microarray analysis to examine the translatome of a panel of peripheral blood (PB) B cells isolated from 34 chronic lymphocytic leukaemia (CLL) patients. We have identified a 'ribosome-related' signature in CLL patients with mRNAs encoding for ribosomal proteins and factors that modify ribosomal RNA, e.g. DKC1 (which encodes dyskerin, a pseudouridine synthase), showing reduced polysomal association and decreased expression of the corresponding proteins. Our data suggest a general impact of dyskerin dysregulation on the translational apparatus in CLL and importantly patients with low dyskerin levels have a significantly shorter period of overall survival following treatment. Thus, translational dysregulation of dyskerin could constitute a mechanism by which the CLL PB B cells acquire an aggressive phenotype and thus have a major role in oncogenesis.

TOPs and their regulation.

Upon cell-cycle arrest or nutrient deprivation, the cellular rate of ribosome production is reduced significantly. In mammalian cells, this effect is achieved in part through a co-ordinated inhibition of RP (ribosomal protein) synthesis. More specifically, translation initiation on RP mRNAs is inhibited. Translational regulation of RP synthesis is dependent on cis-elements within the 5'-UTRs (5'-untranslated regions) of the RP mRNAs. In particular, a highly conserved 5'-TOP (5'-terminal oligopyrimidine tract) appears to play a key role in the regulation of RP mRNA translation. This article explores recent developments in our understanding of the mechanism of TOP mRNA regulation, focusing on upstream signalling pathways and trans-acting factors, and highlighting some interesting observations which have come to light following the recent development of cDNA microarray technology coupled with polysome analysis.

Translation inhibition during the induction of apoptosis: RNA or protein degradation?

The induction of apoptosis leads to a substantial inhibition of protein synthesis. During this process changes to the translation-initiation factors, the ribosome and the cellular level of mRNA have been documented. However, it is by no means clear which of these events are necessary to achieve translational shutdown. In this article, we discuss modifications to the translational apparatus that occur during apoptosis and examine the potential contributions that they make to the inhibition of protein synthesis. Moreover, we present evidence that suggests that a global increase in the rate of mRNA degradation occurs before the caspase-dependent cleavage of initiation factors. Increased mRNA decay is temporally correlated with the shutdown of translation and therefore plays a major role in the inhibition of protein synthesis in apoptotic cells.

Derivation of a structural model for the c-myc IRES.

We have derived a secondary structure model for the c-myc internal ribosome entry segment (IRES) by using information from chemical probing of the c-myc IRES RNA to constrain structure prediction programs. Our data suggest that the IRES is modular in nature, and can be divided into two structural domains linked by a long unstructured region. Both domains are required for full IRES function. Domain 1 is a complex element that contains a GNNRA apical loop and an overlapping double pseudoknot motif that is topologically unique amongst published RNA structures. Domain 2, the smaller of the two, contains an apical AUUU loop. We have located the ribosome landing site and have shown that ribosomes enter in a 16 nt region downstream of the pseudoknots in a situation similar to that observed in several viral IRESs. To test the structure, several key regions of the IRES were mutated and, interestingly, it appears that some of the structural elements that we have identified function to repress c-myc IRES function. This has profound implications for de-regulation of c-myc expression by mutations occurring in the IRES.

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

Mnt is a transcriptional repressor related to the Myc/Mad family of transcription factors. It is expressed in proliferating, resting and differentiating cells and is believed to antagonize the function of Myc. Here we have characterized the major transcription initiation site of the mnt gene. In doing so we noted a remarkable level of sequence conservation between the murine and human 5' untranslated regions. Our experiments revealed that this sequence contains an internal ribosome entry segment (IRES). In addition, we show that sequences at both the 5' and 3' end of the IRES are essential for its function. These findings indicate that mnt can be translated by internal initiation. Such a mechanism may allow efficient Mnt synthesis when cap-dependent translation initiation is reduced.

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

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) (Nanbru et al., 1997; Stoneley et al., 1998) and thus c-myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism (Stoneley et al., 2000). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c-myc and this correlates with a C-T mutation in the c-myc-IRES (Paulin et al., 1996). RNA derived from the mutant IRES displays enhanced binding of protein factors (Paulin et al., 1998). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c-myc-IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c-myc-IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.

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.

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis.

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.

Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment.

The 5' UTR of c -myc mRNA contains an internal ribo-some entry segment (IRES) and consequently, c -myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c -myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c- myc 5' UTR, we demonstrate that both mechanisms can contribute to c- myc protein synthesis. A wide range of cell types are capable of initiating translation of c- myc by internal ribosome entry, albeit with different efficiencies. Moreover, our data suggest that the spectrum of efficiencies observed in these cell types is likely to be due to variation in the cellular concentration of non-canonical translation factors. Interestingly, the c -myc IRES is 7-fold more active than the human rhinovirus 2 (HRV2) IRES and 5-fold more active than the encephalomyocarditis virus (EMCV) IRES. However, the protein requirements for the c -myc IRES must differ significantly from these viral IRESs, since an unidentified nuclear event appears to be a pre-requisite for efficient c -myc IRES-driven initiation.

Translational induction of the c-myc oncogene via activation of the FRAP/TOR signalling pathway.

Previous studies on the regulation of c-myc have focused on the transcriptional control of this proto-oncogene. We have investigated the signalling pathways involved under circumstances in which there is a translational upregulation in the levels of c-myc protein. We have demonstrated an up to tenfold serum-dependent increase of c-myc protein levels in Epstein-Barr virus immortalized B-cell lines 2-4 h after disruption of cellular aggregates, which is not accompanied by an equivalent increase in mRNA. Overall protein synthesis rates only increased threefold suggesting that the c-myc message was being selectively translated. We observed increases in the phosphorylation of p70 and p85 S6 kinases and of initiation factor eIF-4E binding protein 1 (4E-BP1) 1-2 h after stimulation, suggesting activation of the FRAP/TOR signalling pathway. The increased phosphorylation of 4E-BP1 led to a decrease in its association with eIF-4E and an increase in its association with the eIF-4G component of the eIF-4F initiation complex. The signalling inhibitors rapamycin and wortmannin blocked the phosphorylation of 4E-BP1 and abolished the translational component of the c-myc response. Our data suggest that dissociation of eIF-4E from 4E-BP1, leading to an increase in the formation of the eIF-4F initiation complex, relieves the translation repression imposed on the c-myc mRNA by its structured 5'UTR.

C-Myc 5' untranslated region contains an internal ribosome entry segment.

Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5' end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure and in this case ribosomes are directed to the start codon by an internal ribosome entry segment (IRES). Picornaviruses represent the paradigm for this mechanism, but only a few examples exist which show that this mechanism is used by eukaryotic cells. In this report we show data which demonstrate that the 5' UTR of the proto-oncogene c-myc contains an IRES. When a dicistronic reporter vector, with c-myc 5' UTR inserted between the two cistrons, was transfected into both HepG2 and HeLa cells, the translation of the downstream cistron was increased by 50-fold, demonstrating that translational regulation of c-myc is mediated through cap-independent mechanisms. This is the first example of a proto-oncogene regulated in this manner and suggests that aberrant translational regulation of c-myc is likely to play a role in tumorigenesis.