Functional Characterization of the Internal Ribosome Entry Site of eIF4G mRNA

Gan, Weiniu; Celle, Michael La; Rhoads, Robert E.

doi:10.1074/jbc.273.9.5006

Cited by 78 publications

(64 citation statements)

References 47 publications

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“…However, the existence of such a mechanism is recently questioned and argued (Kozak, 2001;Schneider and Kozak, 2001). Furthermore, detailed analysis of the reported IRES element in the 5 0 -UTR sequence of eIF4G (Gan & Rhoads, 1996;Gan et al, 1998) demonstrated that it is in fact a promoter (Han and Zhang, 2002). It was also found that the long 5 0 -UTRs of human Sno and mouse Bad contain promoters, suggesting that promoters in the 5 0 -UTR sequences are more prevalent than anticipated.…”

Section: Discussionmentioning

confidence: 93%

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

et al. 2003

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PTEN tumor suppressor serves as a major negative regulator of survival signaling mediated by PI3 kinase/ AKT/protein kinase B pathway, and is inactivated in various human tumors. Elucidation of mechanisms responsible for PTEN expression is important for providing insight into strategies to control the loss of PTEN expression in human cancers. Although recent studies suggested that p53 and Egr-1 can modulate induced PTEN expression, the mechanism responsible for ubiquitous constitutive expression of PTEN remains elusive. PTEN mRNA contains a highly conserved and GC-rich 5 0 -untranslated region (5 0 -UTR). Recently, it has been shown that the long 5 0 -UTR sequences of several growthregulated mRNAs contain promoters that can generate mRNAs with shorter 5 0 -UTRs. In this paper, we tested whether the 5 0 -UTR sequence of mouse PTEN contains a promoter that is responsible for constitutive expression of PTEN. We found that the long 5 0 -UTR sequence of mouse PTEN severely inhibits translation of PTEN and a heterologous gene firefly luciferase. Deletion of the most 5 0 -UTR sequence would enhance translation efficiency 100-fold. We also showed that the 5 0 -UTR sequence of mouse PTEN does not have an internal ribosome entry site (IRES) that can mediate cap-independent initiation of translation. Instead, we found that the 5 0 -UTR sequence of mouse PTEN contains a strong promoter that drives the production of a transcript with shorter 5 0 -UTRs, which can be translated with higher efficiency. This promoter was mapped to the region between À551 and À220 bases upstream of the translation start codon. Cotransfection analysis using Drosophila SL2 cells showed that Sp1 is one of the major transcription factors that can constitutively activate this promoter. Two endogenous PTEN transcripts with 5 0 -UTRs of 193 and 109 bases were found in DU145 and H226 cell lines. Based on these observations, we conclude that the PTEN expression may be regulated at both transcriptional and translational levels, and that the 5 0 -UTR sequence of PTEN contains a promoter that is responsible for constitutive PTEN expression.

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Section: Discussionmentioning

confidence: 93%

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

et al. 2003

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“…20 Unlike the MYC and BIP IRESes, however, the EIF4G mRNA is structurally similar to the EMCV IRES, and contains cryptic AUG start sites and a functional PPT that is required for activity. 9,21 However, unlike the EMCV IRES, the PPT of the EIF4G IRES cannot be replaced by purines. 9 Another feature that distinguishes the EIF4G IRES from the EMCV IRES is the ability of EIF4G to initiate translation when the AUG start is variably spaced from the PPT, 9 whereas the spacing between the PPT and the initiator AUG in the EMCV IRES is critical for downstream gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…9,21 However, unlike the EMCV IRES, the PPT of the EIF4G IRES cannot be replaced by purines. 9 Another feature that distinguishes the EIF4G IRES from the EMCV IRES is the ability of EIF4G to initiate translation when the AUG start is variably spaced from the PPT, 9 whereas the spacing between the PPT and the initiator AUG in the EMCV IRES is critical for downstream gene expression. 22,23 These features of the EIF4G IRES could perhaps account for its superior efficiency in mediating downstream gene expression.…”

Section: Discussionmentioning

confidence: 99%

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes

2002

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“…We evaluated six putative cellular IRES sequences in this system. These are derived from the 5Ј UTRs of the BiP (12), eIF4G (13), NRF (14), Rbm3 (15), VEGF (16), and XIAP (17) mRNAs. The VEGF sequence used is a variant, termed SP163, reported to exhibit greater activity than the native 5Ј UTR (16).…”

Section: Six Putative Cellular Iress Exhibit Little Activity In a Dicmentioning

confidence: 99%

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

Baranick¹,

Lemp²,

Nagashima³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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....

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Functional Characterization of the Internal Ribosome Entry Site of eIF4G mRNA

Cited by 78 publications

References 47 publications

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

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

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes

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

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