Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

SUMMARY Upstream open reading frames (uORFs) are established as cis -acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9 -associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.