Characterisation of translated downstream Open Reading Frames in human cells

Abstract

This study delves into the characterisation of translated downstream Open Reading Frames (dORFs) in human cells, exploring their role in post-transcriptional gene regulation. dORFs are small ORFs found in the 3’UTR region of an mRNA and have been shown to enhance the expression of its associated main ORF when it is translated in human cells and zebrafish. dORFs as a novel post-transcriptional regulator that works contrary to upstream ORFs (uORF) was established fairly recently. Moreover, we also know that although the presence of dORFs is conserved, the amino acid sequence is not in Orthologous genes in humans and zebrafish, suggesting evolutionary conservation of dORFs. However, how the various structural aspects like dORF length, the 3’UTR length and the iUTR length affect its enhancement activity has not been known until now. Employing a fluorescent reporter expression system, we characterised dORFs to be able to propose how its functional components affect the enhancement ability of the associated canonical open reading frame. Our results demonstrate that the length of dORFs plays a crucial role in modulating translation efficiency, with the presence of an optimal length that exhibits greater enhancement of main ORF expression. We also found that dORFs shorter than 6AA cannot enhance the translation of the main ORF. Additionally, we found the influence of 3’UTR and internal UTR (iUTR) length on dORF-mediated translation enhancement, revealing a dependency on iUTR length but not 3’UTR length for optimal dORF function. With elongated iUTRs, the dORFs lose their capability to enhance the expression of the canonical ORF. These findings provide novel insights into the molecular mechanisms governing post-transcriptional gene regulation, highlighting the diverse roles of dORFs in fine-tuning protein expression levels. Our study lays the groundwork for further investigations into the functional significance of dORFs and their implications for cellular physiology and disease pathogenesis.