Translation initiation mediated by RNA looping

Abstract

Eukaryotic translation initiation commences at the initiation codon near the 5' end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m(7)G cap and/or poly(A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3' UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3' UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a cap-independent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through "RNA looping." The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.

Keywords: RNA looping; eukaryotic mRNA; ribosome scanning; translation initiation.

Fig. 1.

eIF4G tethered at the 3′ UTR of mRNA augments translation of an upstream reporter gene. (A) FLuc represents a reporter RNA containing the firefly luciferase gene as a reporter. MS2-binding sites (6 or 24 copies) were inserted into the reporter RNA to generate FLuc MS2 × 6 and FLuc MS2 × 24, respectively. A stable stem-loop was inserted downstream of the stop codon of reporter RNAs, FLuc and FLuc MS2 × 24, to generate FLuc 3′SL and FLuc 3′SL MS2 × 24, respectively. (B) Schematic diagram of MS2 fusion proteins. (C) The translation efficiencies of FLuc (lanes 1–3), FLuc MS2 × 6 (lanes 4–6), and FLuc MS2 × 24 (lanes 7–9) were determined by measuring firefly luciferase activity in cells expressing MS2-GFP (lanes 1, 4, and 7), MS2-GFP-β-galactosidase (lanes 2, 5, and 8), and MS2-GFP-eIF4G (lanes 3, 6, and 9). Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars reflect SD in three independent experiments. *P < 0.025 compared with lane 3; **P < 9 × 10⁻⁶ compared with lane 3; ***P < 0.005 compared with lane 12. (D) Schematic diagram of reporter mRNAs used in E. Each reporter contains β-globin leader (G-cap), HCV IRES (HCV), EMCV IRES (EMCV), or CrPV IRES (CrPV) at the 5′ UTR. m⁷G-capped reporter RNA containing the Renilla luciferase gene served as a control for mRNA transfection efficiency. (E) Luciferase activity was measured using the extracts from cells transfected with reporters containing m⁷G-capped β-globin leader sequence (lanes 1–6), HCV IRES (lanes 7–12), EMCV IRES (lanes 13–18), or CrPV IRES (lanes 19–24). Ratios of firefly luciferase activity to Renilla luciferase activity were normalized to the ratios obtained from cells transfected with effecter MS2-GFP (lanes 1, 4, 7, 10, 13, 16, 19, and 22). *P < 9.8 × 10⁻⁵ compared with lane 3; **P < 0.003 compared with lane 9; ***P < 0.01 compared with lane 15 (EMCV IRES) or lane 21 (CrPV IRES).

Fig. 2.

5′ end-independent translational activation by eIF4G tethered downstream of a reporter gene. (A) Dual reporters contain Renilla luciferase gene followed by firefly luciferase gene. Twenty-four copies of MS2 sequence exist in reporter mRNA RF MS2 × 24. (B and C) Translation efficiencies from m⁷G-capped (G-RF and G-RF MS2 × 24) and A-capped (A-RF and A-RF MS2 × 24) dual reporters shown in A. Renilla and firefly luciferase activities in each set were normalized to the β-galactosidase activity from a plasmid cotransfected as a transfection efficiency control. Normalized Renilla luciferase activity (B) or firefly luciferase activity (C) of reporter without an MS2 sequence from cells overproducing MS2-GFP proteins were set to 1 (lane 1 for lanes 1–6 and lane 7 for lanes 7–12). (D) Translation efficiencies from m⁷G-capped dual reporters harboring a stable stem-loop (5′ SL RF and 5′ SL RF MS2 × 24). *P < 0.012 compared with lane 3; **P < 0.04 compared with lane 9; ***P < 0.025 compared with lane 9.

Fig. 3.

EMCV IRES stimulates translation of upstream genes. (A) Reporters contain Renilla luciferase followed by WT or mt EMCV IRES with two different kinds of 5′ UTRs. (B) A-capped reporters with or without a stable stem-loop were subjected to in vitro translation with nuclease-untreated RRLs in the presence of 150 mM KCl. Relative translation efficiencies of the reporters harboring a vector sequence (lanes 1–6) or 15 copies of β-globin leader (lanes 7–12) at the 5′ UTR are depicted. The Rluc activity of reporter without EMCV IRES was set to 1 for comparison.

Fig. 4.

5′ end-independent translation enhancement by the EMCV IRES residing downstream of a gene. (A) Dual reporters contain firefly luciferase gene (Fluc) followed by Renilla luciferase gene (Rluc). WT or mt EMCV IRES resides at the 3′ UTR of reporters. (B and C) A-capped reporters were subjected to in vitro translation with nuclease-untreated RRLs in the presence of 150 mM KCl. 5′ end-dependent (Fluc, lanes 1–3 in B and C) and 5′ end-independent (Rluc, lanes 4–6 in B and C) translations were measured and are depicted in graphs. Firefly and Renilla luciferase activities of FR reporter without EMCV IRES were set to 1 for comparison.

Fig. 5.

Comparison of the theoretically calculated probability of collision of two objects on a string with the experimentally observed translation efficiencies of mRNAs with various 5′ UTR lengths. (A) Relative translation efficiencies of mRNAs harboring 5′ UTRs of various lengths. Reporter DNAs, kindly provided by Dr. Vincent Mauro, The Scripps Research Institute, La Jolla, CA, were transfected into HEK293T cells, and luciferase activity was measured at 24 h after transfection. Similar data were reported previously by Chappell et al. (9). Error bars indicate the SD in three independent experiments. (B) Graph depicting the relationship between the local concentration, jM, and the distance, n (number of Kuhn segments), predicted by the equation in Fig. S7B. Because we cannot perform the experiments required to obtain the parameter d, we used d = 0 in the calculation (19). The peak of the graph occurred at n = 1.62.