Fig 1.
JUN translation is regulated by 5’ UTR sequence and structural elements.
(A) Depiction of the full JUN 5’ UTR. The locations of the 208-nt region studied by SHAPE (SHAPE) and the eIF3-interacting stem loop (SL) are marked, along with the nucleotides involved in each region. (B) Secondary structure of the 208-nt region in the JUN 5’ UTR mapped by SHAPE is shown. Nucleotides are numbered according to their position in the 5′ UTR. Mutant JUN 5′ UTR mRNA constructs and their corresponding mutations are described in their associated tables. (C) Luminescence measured from HEK293T cells transfected with the JUN 5′ UTR reporter mRNAs expressing Nanoluciferase (Nluc). Translation was assessed using a dual-luciferase assay and normalized to a control mRNA harboring an HBB 5′ UTR and a Firefly luciferase (Fluc) CDS. Nluc/Fluc ratios were normalized to the WT JUN 5′ UTR, set as 100%. Technical triplicates for each biological replicate, and a total of at least three biological replicates were taken for each measurement. P values determined using a one-sample t test versus a hypothetical value of 100 are shown as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The mean value of the replicates and standard error of the mean are shown.
Fig 2.
JUN is highly sensitive to RocA treatment.
(A) HEK293T cells co-transfected with JUN 5′ UTR and Nluc CDS reporter mRNAs (WT, ΔSL or mutant G, Fig 1) and with an HBB 5’ UTR and Fluc mRNA as an internal control, were treated with increasing concentrations of RocA (+RocA) or DMSO control (+DMSO) 3 hours post-transfection, as previously reported [33]. An mRNA with the HBB 5′ UTR and Nluc CDS mRNA was also used as a RocA-insensitive control. Translation was assessed using a dual-luciferase assay as in Fig 1. Nluc/Fluc measurements were normalized to the corresponding untreated condition (0 nM RocA) and reported as a percentage of this measurement. (B) The location of polypurine (GAA(G/A)) sequences in the JUN 5′ UTR are indicated with yellow lines. Each of these 11 sequences was mutated to CAAC. (C) Luminescence of HEK293T cells transfected with JUN 5′ UTR and Nluc CDS reporter mRNAs (WT, ΔSL, CAAC or CAAC + ΔSL), together with the HBB 5′ UTR and Fluc CDS mRNA control. Transfected cells were treated with 300 nM RocA (+RocA) or DMSO (+DMSO) 3 hours post-transfection. Translation was assessed using a dual-luciferase assay as in Fig 1, and Nluc/Fluc measurements were normalized to the WT JUN 5′ UTR and Nluc CDS +DMSO measurements, reported as percentages. (D) Luminescence from in vitro translation reactions using the JUN 5′ UTR and Nluc CDS reporter mRNAs (WT, ΔSL, CAAC or CAAC + ΔSL). Reactions were treated with 300 nM RocA (+RocA) or DMSO (+DMSO). Luminescence values of each mutant were normalized to the WT JUN 5′ UTR and Nluc CDS +DMSO measurements and reported as percentages. In panels A, C, and D, technical triplicates for each biological replicate, and a total of at least three biological replicates were taken for each measurement. P values determined using a one-sample t test versus a hypothetical value of 100 are shown as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The mean value of the replicates and standard error of the mean are shown.
Fig 3.
Two start codons contribute to JUN translation.
(A) Diagram depicting the ideal Kozak context for a generic open reading frame (A/GnnAUGG). Below, diagrams depicting each of the JUN start codons (AUG) and their translational contexts. (B) Diagram depicting JUN mRNA reporter constructs, with their corresponding mutations in each of the JUN start codons and their translational contexts. The constructs contained the full JUN 5′ UTR sequence along with the first 51 nucleotides of the JUN CDS, upstream of the full Nluc CDS. (C) Luminescence from HEK293T cells transfected with JUN 5′ UTR and 51nt JUN CDS and Nluc CDS reporter mRNAs (WT, ΔuAUG, ΔmAUG, s-uAUG, S-uAUG or S-uAUG W-mAUG), together with an HBB 5′ UTR and Fluc CDS control, assessed using a dual-luciferase assay as in Fig 1. Nluc/Fluc measurements of each mutant were normalized to the WT JUN 5′ UTR and 51nt JUN CDS and Nluc CDS measurements and reported as percentages. Technical triplicates for each biological replicate, and a total of at least three biological replicates were taken for each measurement. P values determined using a one-sample t test versus a hypothetical value of 100 are shown as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The mean value of the replicates and standard error of the mean are shown. (D) Sequence logo depicting the conservation of the 19 nucleotide JUN sequence spanning both start codons and their translational context amongst 100 species. (E) Sequence logo depicting the conservation of the 5 amino acid JUN sequence containing both start codon methionines amongst 100 species. (F) Phylogenetic tree depicting the conservation of both JUN AUGs amongst 100 species. Species with both the JUN uAUG and the JUN mAUG are depicted with blue branches, while species with only one JUN mAUG are depicted in orange.
Fig 4.
JUN translation regulation is mediated by 5’ UTR features and multiple translation initiation factors.
(A) Diagram showing all JUN regions investigated in this study. (B) Diagram depicting different contributors to JUN translation regulation. These factors include: JUN mRNA secondary structure (depicted as a stem loop in the 5′ UTR), JUN sequences (such as the GAA(A/G) polypurine sequences depicted in yellow), JUN AUGs (depicted in orange), and initiation factors (such as eIF3, eIF4A, and potentially eIF1 and eIF5). EIF3 (depicted in pink) interacts with structured regions of the JUN 5’ UTR. EIF4A (depicted in yellow) interacts with GAA(A/G) sequences (black arrow) and with other unknown sequences (gray arrows) in the JUN 5’ UTR. EIF1 (depicted in brown) and eIF5 (depicted in orange) may play roles in JUN start codon selection.