Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

Abstract

As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5 untranslated region (5-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBP␤. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.

Regulation of constitutive expression of mouse PTEN by the 5′-untranslated region

“…The above results suggest that the 5 0 -UTR of PTEN may (1) contain an IRES activity that enhances the translation of firefly LUC from the dicistronic mRNA by internal initiation (for reviews see Pestova et al (2001) and Sachs (2000)), (2) contain a promoter that directs transcription of the firefly LUC gene (Han and Zhang, 2002), and/or (3) contain a splicing acceptor site, that creates a splicing variant with only the second cistron of the firefly LUC gene. To distinguish between these possibilities, we generated dicistronic RNAs in vitro from the dicistronic constructs and used them to program translation both in HeLa cells and in RRL.…”

“…For this purpose, we used a promoterless dicistronic assay as described previously (Han and Zhang, 2002) by simply removing the unique SV40 promoter together with the intron sequence from the pRF-based dicistronic constructs. These promoterless dicistronic constructs ( Figure 6a) were then transfected into HeLa cells for determination of both Renilla and firefly LUC activities ( Figure 6b).…”

“…Recently, we proposed that promoters may exist in the long 5 0 -UTR sequences of genes related to cell growth (Han and Zhang, 2002). In this study, we attempted to address this hypothesis by studying the regulation mechanism of PTEN expression.…”

“…It is possible that PTEN mRNAs use other mechanisms such as internal ribosome entry site (IRES) to initiate translation (for reviews see Pestova et al (2001) and Sachs (2000)). Alternatively, the long 5 0 -UTR of PTEN may contain a promoter or splice site to generate mRNA species with significantly shorter 5 0 -UTRs (Kozak, 2001;Schneider and Kozak, 2001), which can be translated efficiently (Han and Zhang, 2002).…”

Conducting the initiation of protein synthesis: the role of eIF4G

“…However, a recent report has challenged this model showing that protein production driven by the 'IRES' of eIF4G (in the context of bicistronic RNAs) resulted in fact from translation of monocistronic constructs that were generated from a cryptic promoter (Han and Zhang, 2002).…”