The Hidden Language of the 5′ Leader: How uORFs and Introns Shape mORF Translation

The overall aim of my study was to investigate the combinatorial effects of two regulatory elements within the 5′ Leader—upstream open reading frames (uORFs) and introns—on the translational regulation of the main open reading frame (mORF). Although both elements are known to independently influence mORF translation, their interactions remain poorly understood. Addressing this knowledge gap requires the integration of computational and experimental approaches to uncover regulatory mechanisms that may have previously been overlooked. Computational analyses revealed that splice sites within the 5′ Leader exhibit distinct contextual features compared with those in coding regions. The analyses further showed that splice site distribution patterns within ±100 nucleotides of the uORF and mORF start codons were highly similar. Specifically, the peak in splice site density upstream of the mORF start codon was consistent with previous findings reported by Lim et al. (2018; Nucleic Acids Research, 46(9), 4575–4591). In addition, a secondary peak was identified approximately 40–60 nucleotides downstream of the start codon. To experimentally investigate the effects of intron position and uORF presence on mORF translation, a dual-ORF reporter system was developed. Experimental results were consistent with the computational findings, demonstrating that cells transfected with constructs containing introns positioned 50 nucleotides downstream of the uORF exhibited enhanced uORF-to-mORF expression, suggesting that splice site positioning may contribute to translational efficiency. In addition to the experimental assay, a bioinformatic tool was developed to simultaneously demultiplex sequencing samples and retrieve target sequences. This tool, combined with the reporter system, facilitated the identification of optimal uORF start codon sequence contexts that most strongly repress mORF translation. Together, this work provides both computational and experimental frameworks for studying translational regulation at high resolution. The findings highlight the importance of intron positioning and demonstrate that regulatory elements should be considered in combination rather than in isolation when investigating mechanisms governing gene expression.