Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors.

During apoptosis, there is a reduction in translation initiation caused by caspase cleavage of several of the factors required for the cap-dependent scanning mechanism. Under these circumstances, many proteins that are required for apoptosis are instead translated by the alternative method of internal ribosome entry. This mechanism requires the formation of a complex RNA structural element and in the presence of internal ribosome entry segment (IRES)-trans-acting factors (ITAFs), the ribosome is recruited to the RNA. The interactions of several ITAFs with IRESs have been investigated in detail, and several mechanisms of action have been noted, including acting as chaperones, stabilising and remodelling the RNA structure. Structural remodelling by PTB in particular will be discussed, and how this protein is able to facilitate recruitment of the ribosome to several IRESs by causing previously occluded sites to become more accessible.

An internal ribosome entry site in the 5' untranslated region of epidermal growth factor receptor allows hypoxic expression.

The expression of epidermal growth factor receptor (EGFR/ERBB1/HER1) is implicated in the progress of numerous cancers, a feature that has been exploited in the development of EGFR antibodies and EGFR tyrosine kinase inhibitors as anti-cancer drugs. However, EGFR also has important normal cellular functions, leading to serious side effects when EGFR is inhibited. One damaging characteristic of many oncogenes is the ability to be expressed in the hypoxic conditions associated with the tumour interior. It has previously been demonstrated that expression of EGFR is maintained in hypoxic conditions via an unknown mechanism of translational control, despite global translation rates generally being attenuated under hypoxic conditions. In this report, we demonstrate that the human EGFR 5' untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an internal ribosome entry site (IRES). We show that this effect is not due to either cryptic promoter activity or splicing events. We have investigated the requirement of the EGFR IRES for eukaryotic initiation factor 4A (eIF4A), which is an RNA helicase responsible for processing RNA secondary structure as part of translation initiation. Treatment with hippuristanol (a potent inhibitor of eIF4A) caused a decrease in EGFR 5' UTR-driven reporter activity and also a reduction in EGFR protein level. Importantly, we show that expression of a reporter gene under the control of the EGFR IRES is maintained under hypoxic conditions despite a fall in global translation rates.

Removal of canal obstructions with the Endo Extractor.

This report describes a series of clinical cases demonstrating the use of the Endo Extractor to remove canal obstructions. Each of the obstructions was removed with the new technique after conventional methods had failed. Preoperative, working, and postoperative radiographs are shown.

Upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of PTB-1 and YB-1.

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) and c-myc translation can therefore be initiated by internal ribosome entry as well as by cap-dependent mechanisms. It has been shown previously that in patients with multiple myeloma (MM) and in MM-derived cell lines there is a C to T mutation in the c-myc IRES that increases IRES activity and the corresponding synthesis of c-myc protein although it is not fully understood how this occurs. Our data show that two recently identified c-myc IRES trans-acting factors, Y-box binding protein 1 (YB-1) and polypyrimidine tract-binding protein 1 (PTB-1), bind more strongly (approximately 3.5- and 2-fold respectively) to the mutated version of the c-myc IRES and in vitro these proteins exert their effect synergistically to stimulate IRES activity of the mutant IRES 4.5-fold more than the wild-type version. Importantly, we show that there is a strong correlation between the expression of PTB-1, YB-1 and c-myc in MM-derived cell lines, suggesting that by reducing either PTB-1 or YB-1 protein levels it is possible to decrease c-myc expression and inhibit cell proliferation of MM-derived cell lines.

Regulation of BAG-1 IRES-mediated translation following chemotoxic stress.

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5'-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES-meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.

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.

Investigation of interactions of polypyrimidine tract-binding protein with artificial internal ribosome entry segments.

Most eukaryotic translation initiation is thought to be dependent on the 5'-cap structure of the mRNA. It is becoming apparent, however, that the mRNAs of many genes contain IRESs (internal ribosome entry segments) within the 5'-UTR (5'-untranslated region) that allow ribosomes to initiate translation independently of the 5'-cap. IRESs can enable the expression of these genes under conditions (such as viral infection, cellular stress and apoptosis) when cap-dependent translation initiation is compromised, and also provide a target for regulation of gene expression. Recent results from our laboratory and others suggest that 10% of mRNAs (approximately 4000 genes) use this mechanism to initiate translation. One of the central goals of those working in the field of translation is to identify the sequence motif(s) and proteins that are required for internal ribosome entry. We have identified recently a unique PTB (polypyrimidine tract-binding protein) motif (CCU)n that is present in a large subset of cellular IRESs, and the results suggest that PTB itself is involved either directly or indirectly in ribosome recruitment. Here, we describe further investigations of PTB with artificial sequences that harbour this motif.