Neuronal stop-codon readthrough is associated with ribosome pausing and appends lineage-restricted extensions that can alter protein localization in Drosophila

Abstract

Stop-codon readthrough (RT) diversifies proteomes and is particularly prominent in neurons. However, the regulatory logic that specifies neuronal RT and the structural and cellular consequences of the resulting C-terminal protein extensions remain poorly understood. Here we leverage neuron-specific ribosome profiling datasets in Drosophila to construct an in vivo atlas of 163 neuronal RT transcripts. Sequence-based modeling distinguished RT from non-RT transcripts and highlighted an extended post-stop region enriched for stable predicted RNA structures. Beyond these cis-associated features, ribosome-footprint analysis revealed pronounced stop-codon pausing on RT transcripts, accompanied by upstream periodic peaks consistent with ribosome queuing. At the protein level, neuronal RT appended polypeptides enriched in polar residues and intrinsically disordered regions (IDRs). Evolutionary comparisons suggested that most analyzed extensions show shallower phylogenetic conservation than their associated conventional proteins, whereas in two genes a stop-codon appears to have been gained within ancestral coding sequences. Finally, an in vivo dual-color reporter showed that RT of the RNA-binding protein Bru3 alters localization from the nucleus to cytoplasmic granules. Together, our results suggest that neuronal RT is associated with structured post-stop RNA regions that reshape termination dynamics and produce lineage-restricted, IDR-rich C-terminal protein extensions with distinct subcellular localization.