Journal: The EMBO Journal

Article Title: Inhibitors of eIF1A-ribosome interaction unveil uORF-dependent regulation of translation initiation and antitumor and antiviral effects

doi: 10.1038/s44318-025-00449-6

Figure Lengend Snippet: ( A ) HEK293 cells were treated with DMSO or 1Ais for 3 h and subjected to polysome profiling by fractionating cell lysates by centrifugation on a sucrose density gradient (10–50%) followed by OD measurement (254 nm) of each fraction. The quantified polysomes to monosomes ratio is shown on the right. The bars represent the mean, n = 2 independent biological replicates. ( B – I ) The effect of translation inhibition by 1Ais on global translation using Ribo-seq. HEK293T cells were treated with DMSO or 1Ai-3638 and 1Ai-8214 for 3 h then cycloheximide addition, followed by preparation of ribosome footprinting libraries, deep sequencing and analysis according to standard RF protocol. In parallel the same samples were also subjected to RNA-seq for total mRNA level measurements. ( B ) Relative mRNA abundance after 1Ais or DMSO treatment. The bars represent the mean, n = 2–3 independent biological replicates. ( C ) Metagene analysis. The p-site in each ribosome footprint was located and the ribosomal occupancy of each area on the mRNA was calculated and normalized to mRNA total counts. Data is normalized to the TIS peak. ( D ) A list summarizing the number of genes that are differentially translated relative to DMSO control (relative TE > 1.5 or relative TE < 0.5). ( E ) Venn-diagram representing the intersection between genes downregulated by 1Ai-3638 and 8214. The significance of the overlap is P < 10 −5 and was calculated by Randomization test. ( F ) Analysis of the nucleotide context of the annotated start codon of unaffected and 1Ai-3638 and 1Ai-8214 downregulated genes and the overlapping downregulated gene set. Black arrows indicate affected nucleotides. ( G ) The frequencies of the C + 5 and A/G-3 in 1Ais unaffected and downregulated mRNAs; * P < 0.05, * P < 0.01, Randomization test. ( H ) Cryo-EM structure of yeast eIF1A, including its NTT in the entry channel of the ribosome (PDB:6ZMW). The distances were measured by Pymol. ( I ) Three barcoded firefly reporter mutants were generated (scheme on the top) based on the IRF7 5'UTR (IRF7/Kozak, IRF7-C5, IRF7/Kozak-C5). These constructs were co-transfected to HEK293T cells and, 6 h after transfection, incubated with either DMSO, 1Ai-3638, or 1Ai-8214 (20 µM). Cell lysates were fractionated by sucrose gradient sedimentation. RNA was extracted from the polysomes and total fractions followed by DNase I treatment and RT-qPCR using primers specific for each barcode. Data is presented as the ratio of polysomes vs total RNA abundance after 1Ai treatment relative to DMSO; n = 3 independent biological replicates, * P < 0.05, t test, error bars represent SEM. ( J ) 5'UTR length distribution among downregulated and unaffected genes after 1Ais treatment in which the center = median, box ends = interquartile range (IQR), bottom whisker = minimum. Unchanged are 10,569, 3638 down 246 and 8214 down 325 genes. ( K ) The number of uORFs per gene among downregulated and unaffected genes. The presence of uORFs was determined based on previous RF data (Sehrawat et al, ). ( L ) A scheme of firefly luciferase reporters that include the reference 5'UTR (Fig. ), which was altered to have an uORF starting 46nt (short reinitiation, SRI) or 95 nt (Long reinitiation, LRI) or an AUG 46nt before the main ORF without an in-frame stop codon to create a reporter with leaky scanning (LS). ( M ) The reference and uORF-bearing reporters were transfected to HEK293T cells together with RL, which served to normalize translation efficiency. The reporter activities are represented as firefly vs RL; n ≥3 independent biological replicates, error bars represent SEM. * P < 0.05, ** P < 0.01, Student t test. ( N ) Firefly Reporters ( J ) were transfected to HEK293T cells together with normalizing RL. Six hours after transfection, DMSO or 1Ais at 20uM (except 1Ai-5175 at 5uM) were added for overnight incubation. Results are represented as firefly vs. RL normalized to DMSO in each biological replicate; n = 4, error bars represent SEM. * P < 0.05, ** P < 0.01, Student t test. .

Article Snippet: In all, 24 h after transfection, cell lysates were prepared and ribosomal complexes were separated by sucrose gradient sedimentation. mRNA was isolated (Direct-zol RNA Mini prep, #R2062, ZYMO research) from the total fraction (before sucrose sedimentation) and the polysomes fractions. cDNA was prepared by the High-Capacity cDNA RT kit (#4368814, ThermoFisher) and then quantified by qPCR using barcoded primers.

Techniques: Centrifugation, Inhibition, Footprinting, Sequencing, RNA Sequencing, Control, Cryo-EM Sample Prep, Generated, Construct, Transfection, Incubation, Sedimentation, Quantitative RT-PCR, Whisker Assay, Luciferase