Leaky scanning translates a conserved ORF in the 3′UTR of RPL36A and regulates the expression of ribosomal protein L36a.

The 3′UTRs of eukaryotic mRNAs are major hubs of post-transcriptional regulation that govern the spatial and temporal regulation of gene expression. Although 3′UTRs were long considered non-coding, recent studies have revealed translation within downstream open reading frames (dORFs) located in 3′UTRs. In contrast to upstream ORFs (uORFs) in 5′UTRs, the mechanisms governing dORF translation and their functional significance remain poorly understood. Here, we identify and characterize a dORF within the 3′UTR of RPL36A, which encodes ribosomal protein L36a. Analysis of ribosome profiling datasets revealed ribosome footprints aligned with the dORF reading frame across multiple human tissues and cell lines. Conservation analysis together with luciferase- and GFP-based reporter assays further supported active translation of the dORF. Using strategically placed RNA hairpin structures and mutational analyses, we show that dORF translation is consistent with ribosomal leaky scanning. Disruption of dORF translation, either by mutating its start codon in exogenous constructs or by CRISPR-mediated deletion of the endogenous dORF-containing region, reduced RPL36A mRNA and protein levels and resulted in impaired global translation. We further identified a miR-5701-binding site within the dORF region and provide evidence that dORF translation antagonizes miR-5701-mediated repression of RPL36A. Finally, we identify a similar dORF-associated regulatory mechanism in RPS3, which encodes ribosomal protein S3. Together, our findings uncover dORF translation as a regulatory mechanism controlling ribosomal protein expression.