Integrative Analysis Identifies a Conserved Start–Stop Element and Downstream RNA G-Quadruplex That Repress DUSP4 Translation

Abstract

Upstream start–stop elements represent an emerging class of translational regulators that can strongly repress downstream protein synthesis. However, the contribution of adjacent RNA secondary structures to their regulatory activity remains poorly understood. Here, we investigated the potential interplay between start–stop elements and downstream RNA G-quadruplexes (rG4s) in human mRNA 5′ untranslated regions (5′-UTRs). By integrating ribosome profiling data with experimentally supported rG4 annotations, we identified candidate start–stop elements associated with nearby rG4-prone sequences. Among these, DUSP4 was selected for further investigation based on its pronounced ribosome occupancy peak at the start–stop element, the proximity of a downstream G-rich motif, and evolutionary conservation of the start–stop element and its context. Using fluorescence spectroscopy, circular dichroism, UV melting, and NMR experiments, we demonstrated that the downstream G-rich motif forms a stable, potassium-dependent, three-layered rG4 in vitro. Functional analysis in HEK293T cells revealed that disruption of the start–stop element significantly increased reporter expression in both plasmid- and mRNA-based systems. Mutations in the rG4-prone sequence enhanced reporter activity in the plasmid system. Together, these findings identify a functional start–stop element in the DUSP4 5′-UTR and a closely positioned, structurally characterized rG4-prone motif, suggesting a potential additional layer of post-transcriptional regulation.

Abstract as supplied by the indexing source. © the authors / publisher.