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. 2004 Aug 15;18(16):1997-2009.
doi: 10.1101/gad.1212504.

Tethering of eIF4G to adenoviral mRNAs by viral 100k protein drives ribosome shunting

Qiaoran Xi  1 , Rafael Cuesta, Robert J Schneider
Affiliations

Tethering of eIF4G to adenoviral mRNAs by viral 100k protein drives ribosome shunting

Qiaoran Xi et al. Genes Dev. .

Abstract

Although most mRNAs initiate translation by 5' ribosome scanning, some small fraction of mammalian and viral mRNAs utilize either of two alternate mechanisms, known as internal ribosome entry and ribosome shunting. Ribosome shunting is a poorly understood form of initiation in which 40S ribosome subunits are loaded onto mRNA through interactions with the m7GTP cap, but then bypass large segments of the mRNA as directed by cis-acting RNA shunting elements and trans-acting protein factors. Here, we describe the molecular mechanism by which ribosome shunting occurs with high efficiency on adenovirus late mRNAs. We show that the viral 100k protein possesses a selective binding element for the 5' noncoding region (5'NCR) of viral late mRNAs (known as the tripartite leader), forms a complex with initiation factor eIF4G and poly(A)-binding protein (PABP), and strongly and selectively enhances the level of both factors and 40S ribosome subunits on viral mRNAs in polysomes. Mutational and biochemical studies demonstrate that the ability of 100k protein to bind both the tripartite leader and eIF4G are critical to promote a high level of ribosome shunting. A molecular mechanism for ribosome shunting is described by which enhanced binding of eIF4G and possibly PABP with 100k protein, and simultaneous interaction with the tripartite leader 5'NCR, drives 40S ribosome recruitment and initiation on mRNAs.

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Figures

Figure 1.
Figure 1.
Ad late 100k protein specifically facilitates translation by ribosome shunting on the tripartite leader. (A) Diagram of 5′NCR elements used in this study. Shown are the CR3 5′NCR (strongly eIF4F dependent), the wild-type tripartite leader (wt 3LDR), the B202 tripartite leader that translates exclusively by ribosome shunting, and the EMCV IRES-GFP construct. (B) The 293 cells were cotransfected with pIRES-EGFP and plasmids expressing β-galactosidase (β-gal) mRNA containing 3LDR or B202 mutant 5′NCRs. At 18 h posttransfection, cells were mock infected or infected with Addl309 for 30 h, labeled with [35S]methionine for 30 min, β-gal was recovered by immunoprecipitation, resolved by SDS-PAGE and fluorographed. GFP protein was detected by immunoblot using anti-GFP antibodies. mRNA levels were analyzed by Northern blot. A contaminating viral protein is indicated. (C) Cells were transfected as above and infected with 100k mutant Adts1 at 39.5°C (restrictive temperature) for 30 h (late infection), or at 33°C (nonrestrictive temperature) for 60 h (late infection), labeled, and proteins processed as above. (D) The 293 cells were transfected with wild-type or B202 tripartite leader reporters as above, with or without cotransfection of Flag-100k plasmid (controlled by the tripartite leader), infected with Adts1 at 39.5°C for 30 h, and proteins analyzed as described above. Autoradiograms were quantified by densitometry, and typical results of at least three independent experiments were used to calculate mean values and standard deviations reported in the text.
Figure 2.
Figure 2.
Ad 100K protein is sufficient to facilitate ribosome shunting on tripartite leader mRNAs. The 293 cells were cotransfected with plasmids expressing β-gal mRNA containing wild-type (3LDR) or shunting-only (B202) Ad tripartite leader 5′ UTRs or a 5′ UTR (CR3) that is strongly dependent on eIF4F, with Flag, or Flag-100k plasmids (using the tripartite leader) and pIRES-EGFP. (A) At 36 h posttransfection, β-gal mRNA levels were determined by Northern blot hybridization, cells were labeled with [35S]methionine, normalized to GFP and mRNA levels, and β-gal levels determined by immunoprecipitation, SDS-PAGE, and fluorography. (B) The 293 cells were cotransfected with plasmid pIRES-EGFP, vector alone (pFLAG-CM2), or a Flag-100k expression vector (pTL-FLAG100), and increasing amounts of plasmids pCMV-Ad LUC (3LDR) or pCMV-LUC (CR3). At 36 h posttransfection, luciferase activity was measured and normalized to mRNA levels as determined by Northern blotting. Data were quantified as described in the legend for Figure 1.
Figure 3.
Figure 3.
The 100k protein interaction with eIF4G is necessary but not sufficient for ribosome shunting. (A) Diagrammatic representation of wild-type and mutant 100k proteins used in this study. The general RNA-binding domain (RGG box) and the tripartite leader RNA-recognition motif (RRM) are shown. Numbers refer to amino acid positions of 100k protein. (B) The 293 cells were transfected with vectors expressing wild-type Flag-100k protein, Flag-100k N345 mutant protein, or Flag-100k N726 mutant proteins and β-gal mRNAs containing either the wild-type Ad tripartite leader (3LDR) or tripartite leader mutant B202. At 36 h posttransfection, cells were labeled with [35S]methionine, β-gal protein was recovered by immunoprecipitation, resolved by SDS-PAGE, and fluorographed. Wild-type 100k and mutant 100k proteins were detected by immunoblotting with anti-Flag antibody. (C) The 293 cells were cotransfected with vector alone or wild-type Flag-100k protein, Flag-100k N345 mutant protein, or Flag-100k N726 mutant protein expression vector. Endogenous eIF4GI was recovered from equal amounts of lysate by immunoprecipitation, and the associated wild-type or mutant 100k proteins were detected by immunoblotting with anti-Flag antibody. (D) The 293 cells were cotransfected with pIRES-EGFP and plasmids expressing Flag-tagged wild-type (WT) or mutant 100k RRKA protein and β-gal mRNA. (Top) Immunoprecipitation of endogenous eIF4G and immunoblot of eIF4G and associated Flag-100k proteins, or preimmune [PI] sera. (Bottom) Immunoblot analysis of levels of proteins in total cell lysates are shown for Flag-100k proteins and controls (eIF4A and GFP). (β-gal) Cells were labeled with [35S]methionine for 30 min and β-gal translation rates were determined by immunoprecipitation of β-gal protein (normalized to GFP and RNA levels) and SDS-PAGE/fluorography. (E) Cells were transfected with wild-type or 100kRRKA protein expression vectors expressing luciferase (luc) mRNA containing the B202 Ad tripartite leader. Luciferase activity was measured and translation rates were normalized to GFP protein and luciferase mRNA levels.
Figure 4.
Figure 4.
The 100k protein enhances eIF4G/PABP levels on polysomes containing Ad tripartite leader mRNAs. (A) The 293 cells were transfected with plasmids expressing β-gal mRNA containing the wild-type Ad tripartite leader 5′NCR (3LDR) or the eIF4F-dependent 5′NCR (CR3) and vector or plasmid expressing Flag-100k protein (pFlag-100k). At 36 h posttransfection, cells were fractionated and total lysate, monosome/polysome complexes, and postpolysome supernatants were isolated as described (Morley and Hershey 1990). (A) Equal amounts of total cell lysates, the monosome/polysome fraction, and the postpolysome supernatants were resolved by SDS-PAGE. Proteins were detected by immunoblot using specific antibodies as shown. (B) Equal amounts of monosome/polysome-associated proteins were resolved by SDS-PAGE. Proteins were detected by immunoblot analysis using specific antibodies as shown. (C) RNA was isolated from the monosome/polysome complexes isolated above and detected by Northern blot analysis. (D) Monosome/polysome fractions were prepared in the absence or presence of 25 mM EDTA to disrupt ribosome-mRNA interactions. Fractions were subjected to immunoblot analysis for eIF4G. Northern blot analysis was used to detect reporter β-gal mRNA.
Figure 7.
Figure 7.
eIF4G and tripartite leader-specific binding are required for 100k recruitment of eIF4G and PABP into polysomes containing Ad tripartite leader mRNAs. (A) The 293 cells were transfected with plasmids expressing β-gal mRNA containing the wild-type tripartite leader (3LDR) or the eIF4F-dependent 5′NCR (CR3), and plasmids expressing wild-type or mutant 100k proteins (pFlag-100k, pFlag-100kN345, pFlag-100kRRKA). At 36 h posttransfection, monosome/polysomes complexes were isolated, equal amounts of polysome-associated proteins were resolved by SDS-PAGE, and detected by immunoblot analysis using specific antibodies as shown. (B) The 293 cells were transfected with vector alone, plasmids expressing wild-type Flag-100k, or the Flag-100kRRKA mutant, pIRES-EGFP, and tripartite leader β-gal mRNA. Flag-100k association with tripartite leader β-gal mRNA was determined by immunoprecipitation and quantitative RT-PCR. Total mRNA levels were determined by Northern blot analysis.
Figure 5.
Figure 5.
The 100k protein specifically associates with the viral tripartite leader mRNA in vivo. (A) The 293 cells were cotransfected with either vector alone, plasmids expressing Flag-100k protein, truncated 100k protein mutants N726 or N345, and plasmids expressing β-gal mRNA containing either the wild-type tripartite leader 5′NCR (3LDR) or an eIF4F-dependent 5′ NCR (CR3). Wild-type or mutant 100k proteins were recovered from equal amounts of lysate by immunoprecipitation with anti-Flag antibody, mRNA was extracted from the extensively washed immunoprecipitates and identified by quantitative RT-PCR and ethidium bromide agarose gel electrophoresis. Equal amounts of protein lysates were resolved by SDS-PAGE and immunoblotted with specific antisera as shown. (B) Total RNA was extracted from cells and analyzed by Northern blot hybridization analysis. (C) The 293 cells were transfected with vector alone, plasmids expressing wild-type Flag-100k protein, or 100k point mutant Y36F and tripartite leader β-gal mRNA. Flag-100k proteins were immunoprecipitated and analyzed by quantitative RT-PCR as above. eIF4G was immunoprecipitated and immunoblot analysis carried out for eIF4G and Flag-100k protein. Total β-gal mRNA was analyzed by Northern blot. (D) The 293 cells were cotransfected with a plasmid expressing the tripartite leader B202 β-gal mRNA and vector alone, wild-type Flag-100k, or the Y365F 100k mutant and luciferase activity determined. Standard deviations were calculated from at least three independent experiments.
Figure 6.
Figure 6.
The 100k mutant N726 rescues the Adts1 virus to promote ribosome shunting at restrictive temperature. (A) The 293 cells were cotransfected with pIRESEGFP and plasmids expressing β-gal mRNA containing the B202 tripartite leader, Flag-tagged 100k protein or Flag-tagged 100kN726 protein (with the tripartite leader as the 5′NCR). At 18 h posttransfection, cells were infected with Adts1. After 30 h of infection at 39.5°C, cells were labeled with [35S]methionine, β-gal was recovered from equal amounts of lysate by immunoprecipitation, resolved by SDS-PAGE, and fluorographed. Autoradiograms were quantified by densitometry. Other proteins were detected by immunoblot analysis using specific antibodies as shown. Luciferase activity was determined from equal amounts of cell lysates and averaged for three independent experiments.
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