The dicistronic assay for internal ribosome entry site (IRES) activity is the most widely used method for testing putative sequences that may drive cap-independent translation initiation. This assay typically involves the transfection of cells with dicistronic DNA test constructs.

The cellular response to stress involves a rapid inhibition of cap-dependent translation via multiple mechanisms, yet some translation persists. This residual translation may include proteins critical to the cellular stress response. BCL-2 is a key inhibitor of intrinsic apoptotic signaling. Its primary transcript contains a 1.45-kb 5 -untranslated region (UTR) including 10 upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Thus, we hypothesized that this 5 -UTR may contain an internal ribosome entry site (IRES) that facilitates BCL-2 translation, particularly during cell stress. Here we show that the BCL-2 5 -UTR demonstrated IRES activity both when translated in vitro and also when m 7 G-capped and polyadenylated mRNA was transiently transfected into 293T cells. The activity of this IRES in unstressed cells was ϳ6% the strength of the hepatitis C virus IRES but was induced 3-6-fold in a dose-dependent manner following short term treatment with either etoposide or sodium arsenite. Thus, the IRES-mediated translation of BCL-2 may enable the cell to replenish levels of this critical protein during cell stress, when cap-dependent translation is repressed, thereby maintaining the balance between pro-and anti-apoptotic BCL-2 family members in the cell and preventing unwarranted induction of apoptosis.

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5 end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5 sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5 untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

Cellular inhibitor of apoptosis protein 1 (c-IAP1) can regulate apoptosis through its interaction with downstream TNF receptor effectors (TRAF1 and TRAF2), by binding to and inhibiting certain caspases, and by controlling the levels of specific proapoptotic stimuli (e.g., Smac/DIABLO) within the cell. Studies involving the expression of c-IAP1 mRNA and protein in cells and tissues have provided evidence suggesting c-IAP1 expression may be posttranscriptionally controlled. Because the 5-UTR of c-IAP1 mRNA is unusually long, contains multiple upstream AUG codons, and has the potential to form thermodynamically stable secondary structures, we investigated the possibility it contained an internal ribosome entry site (IRES) that may regulate its expression. In the present study, the c-IAP1 5-UTR exhibited IRES activity when dicistronic RNA constructs were translated in rabbit reticulocyte lysate (RRL) and in transiently transfected cells. IRES-mediated translation was similar to that exhibited by the hepatitis C virus IRES but varied significantly in RRL and in HeLa, HepG2, and 293T cells, indicating the c-IAP1 IRES was system and cell type specific. IRES-mediated translation was maintained in mono-and dicistronic constructs in which the UTR was inserted downstream from a stable hairpin that prevented cap-dependent ribosome scanning. In cells, the presence or absence of a methylated cap did not significantly affect the translation of polyadenylated, monocistronic RNAs containing the c-IAP1 5-UTR. IRES-mediated translation was stimulated in transfected cells treated with low doses of pro-apoptotic stimuli (i.e., etoposide and sodium arsenite) that inhibited endogenous cellular translation.

Eukaryotic translation initiation factor 4GI (eIF4GI)is an essential scaffolding protein required to recruit the 43 S complex to the 5-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5-UTR per se. However, the 5-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5 m 7 guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of capdependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.