Splicing mediates the activity of four putative cellular internal ribosome entry sites

Abstract

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5 end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3 splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3 splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.T he vast majority of eukaryotic mRNAs are translated via a mechanism in which the 40S ribosomal subunit engages the mRNA at its methylguanosine-capped 5Ј end (1). Upon associating with the transcript, these subunits are believed then to scan in the 5Ј to 3Ј direction for an appropriately situated AUG at which to begin translation (2, 3). A smaller number of mRNAs are translated by a 5Ј end-and cap-independent mechanism wherein ribosomes are recruited to the transcript at an interior location through an internal ribosome entry site (IRES).IRESs were first discovered in the picornaviruses encephalomyocarditis virus (EMCV) and poliovirus (4, 5). The RNA of these viruses possesses very long 5Ј UTRs bearing many unutilized upstream AUGs (uAUGs) and, unlike cellular mRNA, is uncapped (6). Soon after the identification of picornaviral IRESs, a number of cellular mRNAs were also reported to contain IRESs. To date, at least 85 cellular IRESs have been described (7). The experimental grounds on which proof of most cellular IRESs rest, however, has been the subject of dispute (8-10).A primary criticism of the data presented as establishing the existence of cellular IRESs concerns the plasmid-based dicistronic assay, the standard method of ascertaining IRES activity. In this assay, the candidate sequence is inserted between two reporter genes (5) so that both the upstream and downstream cistron are transcribed on the same RNA. If the test insert causes increased expression of the downstream cistron relative to the upstream cistron, the result is considered evidence for internal ribosome entry. However, the generation of even low levels of monocistronic RNAs from dicistronic constructs has the potential to falsely indicate IRES activity (8, 9, 11). One way that such RNAs could arise is through splicing of the dicistronic transcript due to the presence of a 3Ј splice site (ss) in the test sequence [see supporting information (SI) Fig....

Expression of Truncated Eukaryotic Initiation Factor 3e (eIF3e) Resulting from Integration of Mouse Mammary Tumor Virus (MMTV) Causes a Shift from Cap-dependent to Cap-independent Translation

“…The anti-apoptotic protein XIAP, an important factor for tumor cell survival and resistance to apoptosis after treatment with anti-cancer agents (46 -48), is regulated by a translational mechanism (36). Initially it was thought that this mRNA contained an IRES (36), but a subsequent study showed that a 3Ј splice site was necessary for apparent IRES function (49). The mRNA encoding c-Myc shifts to heavier polysomes under conditions of reduced cap-dependent translation (45), and evidence has been presented that it contains an IRES (37).…”

The Current Status of Vertebrate Cellular mRNA IRESs

“…So this ubiquitous presence of a 5 0 -proximal splice donor site poses a strong risk of mis-splicing to any cryptic 3 0 -splice site present in the putative IRES under test (Fig. 3C), as shown by the finding that when all potential 5 0 -splice sites in the 5 0 -UTR and the upstream cistron (in this case coding for Gaussia luciferase) were inactivated by mutations, the BiP, NRF, VEGF, and XIAP 5 0 -UTRs, as well as the eIF4GI 5 0 -UTR reported by Gan and Rhoads (1996), failed to show significant IRES activity above the "no IRES" control, or the b-globin 5 0 -UTR, although the EMCV IRES positive control yielded high activity (Baranick et al 2008). In the same publication a sensitive assay involving incorporating the IRES and a GFP reporter into a murine leukaemia virus proviral DNA (Fig.…”

“…Indeed it has been shown that in a Krebs-2 in vitro system, or in an RNA transfection assay, an mRNA with the 900 nt (60% GC) LINE-1 5 0 -UTR, is translated by the cap-dependent scanning mechanism at 50% relative efficiency compared to the b-globin 5 0 -UTR (Dmitriev et al 2007). It should also be noted that some long 5 0 -UTRs with apparent IRES activity have subsequently been shown to be incompletely spliced variants with retained introns (Baranick et al 2008), for example the original eIF4GI 5 0 -UTR studied by Gan and Rhoads (1996), in contrast to the eIF4GI 5 0 -UTRs described by Johannes and Sarnow (1998) and Byrd et al (2005).…”

“…Moreover, a database gag-pol 5′ 5′ splice site 3′ env IRES GFP Figure 4. Highly sensitive murine leukaemia virus test for the presence of 3 0 -splice sites in putative IRESs (Baranick et al 2008). The putative IRES and GFP reporter are inserted into the proviral DNA (driven by a CMV promoter) downstream from the env gene.…”

Widespread establishment and regulatory impact of Alu exons in human genes

“…3D), suggesting a potential IRES created by Alu exon inclusion. However, to confirm the identity of the IRES element definitively and to define its exact location in the Alu exon inclusion 5′-UTR requires substantial additional experimental efforts (39,40).…”