Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray

Abstract

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5 end-dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5 noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

Overexpression of the eukaryotic translation initiation factor 4G (eIF4G‐1) in squamous cell lung carcinoma

“…The additional bands of proteins in the Western blot analysis may represent products of degradation of the eIF4G-1 protein. 15 However, there is an overall good correlation between the results from the Western blot analysis and the immunohistochemistry. The immunohistochemic staining method has the advantage of being able to localize the protein, and therefore the cytoplasmic localization of the translational factor eIF4G-1 184 was confirmed.…”

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

“…Polysome profiling has been carried out under a number of conditions when cap-dependent translation has been inhibited including following polioviral infection, 30,31 during mitosis, 32 hypoxia and apoptosis (our unpublished data). In each of these diverse conditions, it has been found that approximately 3% of the messages remain associated with the polysomes including c-myc mRNA.…”

Two internal ribosome entry sites mediate the translation of p53 isoforms

“…All bicistronic constructs had the respective 5 0 UTR sequences cloned between Rluc and Fluc genes, in pCDNA3.1, which does not contain a chimeric intron. p53 5 0 UTR sequences were also cloned downstream of the inactive DEMCV IRES sequence in the intercistronic region of the plasmid pRDDEF (Johannes et al, 1999). The DEMCV sequence was also cloned upstream of Rluc gene in the bicistronic plasmids.…”

“…These results indicated the presence of IRES elements in both p53( þ 39) and (À1) sequences. To rule out the ribosomal read-through of the intercistronic sequences, they were cloned in the plasmid pRDEF, downstream of a stable RNA structure derived from the encephalomyocarditis virus (EMCV) IRES that inhibits ribosomal read-through (Johannes et al, 1999). These bicistronic DNAs, pRDEp53( þ 39)F and pRDEp53(À1)F, produced 13-and 12-fold higher Fluc activity, respectively, than that from the control vector pRDEF (Fig 2B(ii)).…”