Unique translation initiation of mRNAs-containing TISU element

Abstract

Translation Initiator of Short 5' UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5' UTR). Here, we investigated translation initiation from short 5' UTR mRNAs with AUG in various contexts. Reducing 5' UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5' UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.

Figure 1.

(A) The sequences of TISU, strong and weak AUG contexts were cloned DS to a T7 promoter so that their AUG is in frame with the authentic GFP AUG and is distant from the m7G cap by 11, 8 or 5 nt. These constructs were used to synthesize capped mRNAs in vitro. (B) The in vitro synthesized mRNA described in (A) were co-transfected into 293T cells together with luciferase mRNA that serves to normalize transfection efficiency. Translation of the GFP and luciferase proteins were analyzed by immunoblot with GFP antibody and by luciferase assay, respectively. Representative blot of in vivo-translation experiments is shown in the upper panel. Initiation from the upstream AUG (US) produces a protein of 30 KDa and from the DS 27 KDa. Quantified results of three independent transfection experiments are shown at the bottom panel.

Figure 2.

The 48S-ribosomal subunit binds to mRNAs-bearing TISU at the 5′-end with high affinity. (A) A toe-printing assay on mRNAs-bearing AUG within TISU (T) or strong (S) contexts. A schematic representation of the mRNA and the ³²P-labeled primer used for toe-printing procedure is shown in the right. The constructs were in vitro transcribed and annealed with ³²P-labeled primer. RRL was incubated with the non-hydrolyzable GTP analog, GMP-PNP, at 25°C for 10 min, then the mRNA was added for additional 10 min. Subsequently the location of the 48S on the mRNA was analyzed by primer extension as shown in the representative gel. The locations of 48S binding site boundary (∼15 nt DS of the AUG) are marked. A detailed description of the sequence of the analyzed region is shown in Supplementary Figure S3.

Figure 3.

The effect of a moderate secondary structure on the efficiency and fidelity of translation from a strong and TISU AUG contexts located five nt from the m7G cap. (A) Schematic representation of a secondary structure positioned 6 or 15 nt DS of the AUG is shown on the top panel. The constructs with or without secondary structure were in vitro transcribed and then translated in vivo by transfection into 293T cells and translation efficiency was assayed by immunoblot with anti-GFP. Representative immunoblots of three independent experiments is shown. The graphs represent the average ± SD of the intensity of the upstream (30 KDa) and DS (27 KDa) translation site of 3–5 independent experiments (In the case of the strong AUG, the densitometry analysis was done using the blot from the long exposure).

Figure 4.

Translation initiation from TISU is dependent on 7mG cap. (A) mRNAs bearing AUG with either TISU or a strong contexts located either 5 or 11 nt from the 5′-termini were in vitro transcribed in the presence of m⁷GpppG cap or the unmethylated cap-analog ApppG. The mRNAs were translated in vivo by transfection into 293T cells. Cell lysate was prepared 24 hours following transfection and subjected to western blot using anti-GFP. Translation efficiency is shown in the representative blot of three independent experiments. (B) The effect of AUG upstream secondary structure on the efficiency and fidelity of translation from a strong and TISU AUG contexts. The upper-left panel shows a schematic representation of a secondary structure located upstream from the AUG within TISU or strong AUG context. The constructs with or without secondary structure were in vitro transcribed and then translated in vivo by transfection into 293T cells and translation efficiency was assayed by blotting with anti-GFP. Representative immunoblot for the strong and the TISU AUG contexts are shown in the bottom-left panel and the graphs represent the average ± SD of the intensity of the upstream (30 KDa) translation site of four independent experiments. (C) Recognition of TISU's AUG is dependent on the 5′-end of mRNA. The upper panel shows a schematic representation of mRNAs bearing either one or two TISU elements in tandem with the expected protein size translated from each AUG. The mRNAs were transcribed and capped in vitro and then were translated in vivo by transfection into 293T cells. Cell lysate was prepared 24 hours following tranfection and subjected to western blot using anti-GFP (lower panel). The positions of 19 and 37 KDa proteins are shown by arrows.

Figure 5.

The activity of TISU is dependent on amino acid availability. (A) HeLa cells were transfected with in vitro-synthesized GFP–mRNA driven either by TISU or a strong initiation context preceded by a 5-nt long, 5′ UTR. Cells were incubated for 24 h either in full medium (control) or amino acid-free medium (W/O AA) or full medium containing 20 nM rapamycin. The serum used for cell growth was dialyzed prior to use to remove residual amino acids. The cells were harvested and GFP levels were detected by a GFP antibody. Representative western blots are shown and the graph at the bottom represents the average ± SD of the intensity of the upstream (30 KDa) translation site of four independent experiments. (B) Analysis of 4EBP phosphorylation status. Cell lysates from the experiment described in (A) were subjected to western blot with antibodies against total or antibodies specific to the un-phosphorylated form of 4EBP as indicated.

Figure 6.

A. The influence of eIF1 on translation initiation in vitro from short 5′ UTR mRNA and distinct-AUG contexts. (A) schematic representation of the GFP-reporter gene with either TISU or a strong AUG context, both with 11 nucleotides, 5′ UTR. eIF1 was expressed in E. coli, purified and the indicated amounts were added to in vitro-translation reactions with the described constructs. Reactions with TISU and the strong AUG context are indicated at the top. (B) The effect of eIF1 on translation efficiency and accuracy in vivo. The upper panel shows a scheme of the GFP-reporter gene driven either by TISU or by a weak-AUG context, both with short 5′ UTR. The AUG-flanking sequence is shown. These reporters were transfected into HEK293T cells with increasing amounts of eIF1-expression plasmid as indicated, and the translation-initiation site was determined by immunoblot with GFP-specific antibody. US and DS denote upstream and downstream initiation site, respectively. eIF1 expression was analyzed by immunoblot using anti-HA antibody. (C) A graph representing translation directed by TISU or the weak-AUG reporter, GFP, in the absence or presence of low dose of eIF1 plasmid (250 ng), from three independent experiments (average ± SD). The overall translation without eIF1 was set to one. The relative intensity of the upstream translation site is presented by light grey bars and the DS-translation site by dark grey bars. The asterisks denotes statistically significant difference, P < 0.005.

Figure 7.

The effect of a dominant negative mutant of eIF4A on translation. (A) schematic representation of the GFP-reporter genes is shown in the upper-left panel. The first has a long unstructured 5′ UTR, the second has a hair-pin structure within the 5′ UTR and the third has TISU in a context of a short 5′ UTR. HEK293T cells were transfected with these GFP reporters together with increasing amounts of eIF4A-dominant negative mutant (eIF4A-DN) or wild-type eIF4A-expression plasmids as indicated. The amount of expression plasmid was kept constant with the empty expression vector. RL under the CMV promoter was found to be refractory to eIF4A-DN (see Supplementary Figure S6) was used to normalize for transfection efficiency. GFP, eIF4A-DN and eIF4A expression were analyzed by immunoblot in which representative are shown on the upper right section. The asterisk in the eIF4A blot denotes a non-specific band. A graph representing the average of densitometric measurements of three independent transfection experiments is shown in the lower panel.