Fig. 2: Double-stranded RNA element cooperates with initiation codon to activate GATA4 uORF translation.

a Schematic highlighting the key steps of RNA SHAPE workflow used for the human GATA4 uORF-dsRNA-bearing RNA sequence. b RNA SHAPE analysis of human GATA4 uORF-dsRNA-bearing RNA region. A sequencing gel resolved individual nucleotides of the SHAPE assay products. Primers 1 and 2 were used to detect the downstream and upstream dsRNA strands in the GATA4 RNA. Experiments in b were carried out twice, and one representative result is shown. c Densitometric quantification of band intensity corresponding to the structured nature of a given nucleotide using SAFA. The upper inserts represent two strands of the dsRNA. Lower NAI SHAPE reactivity (band intensity) indicates dsRNA structure. Two biological replicates from b were plotted. d The predicted lowest free energy secondary structure of human GATA4 uORF surrounding region based on SHAPE reactivity data using RNAstructure. The resolved region with detectable SHAPE activity is at 252-440 nt. e Left: Schematic of firefly luciferase (FLuc) reporters that include variants of the full-length RNA sequences of human GATA4 mRNA before the start codon of mORF (including uORF-dsRNA). ΔuORF ATG-to-TTG start codon mutation, MM mismatch mutations in dsRNA, Rescue:mismatch and compensatory mutations. Middle: dual luciferase reporter assay with WT and mutants. Following transfection into HEK293T cells, FLuc levels were normalized to a control RLuc reporter. Right: RT-qPCR of FLuc mRNA normalized to ACTB. Data are represented as mean ± SD. **P < 0.01; Statistical significance was confirmed by an unpaired two-tailed Student t test for e (N = 3 biological replicates). Source data are provided as a Source Data file.