Alternative ways to think about cellular internal ribosome entry.
ABSTRACT Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of "cap-dependent" and "IRES-dependent" should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.
- SourceAvailable from: Jian-Ting Zhang[show abstract] [hide abstract]
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/EBPbeta. 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.Molecular and Cellular Biology 12/2002; 22(21):7372-84. · 5.37 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The DKC1 gene encodes a pseudouridine synthase that modifies ribosomal RNA (rRNA). DKC1 is mutated in people with X-linked dyskeratosis congenita (X-DC), a disease characterized by bone marrow failure, skin abnormalities, and increased susceptibility to cancer. How alterations in ribosome modification might lead to cancer and other features of the disease remains unknown. Using an unbiased proteomics strategy, we discovered a specific defect in IRES (internal ribosome entry site)-dependent translation in Dkc1(m) mice and in cells from X-DC patients. This defect results in impaired translation of messenger RNAs containing IRES elements, including those encoding the tumor suppressor p27(Kip1) and the antiapoptotic factors Bcl-xL and XIAP (X-linked Inhibitor of Apoptosis Protein). Moreover, Dkc1(m) ribosomes were unable to direct translation from IRES elements present in viral messenger RNAs. These findings reveal a potential mechanism by which defective ribosome activity leads to disease and cancer.Science 06/2006; 312(5775):902-6. · 31.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.Molecular and Cellular Biology 04/1988; 8(3):1103-12. · 5.37 Impact Factor