Translation of upstream open reading frames in a model of neuronal differentiation

Upstream open reading frames (uORFs) initiate translation within mRNA 5’ leaders,and have the potential to altermain coding sequence(CDS) translationontranscripts in which they reside. Ribosome profiling(RP)studies suggest that translating ribosomes are pervasive within 5’ leadersacross model systems. However, the significance of this observationremains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre),we identify4,954uORFsacross31%of all neuroblastoma transcripts. These uORFspredominantly utilize non-AUG initiationcodonsand exhibit translational efficiencies(TE)comparable to annotated coding regions. Usage of both AUG initiated uORFs and aconservedandconsistently translated subset of non-AUG initiated uORFs correlateswith repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. Withneuronal differentiation, we observedan overall positive correlation between translational shifts in uORF/CDSpairs. However,a subset of transcripts exhibit inverse shifts in translation of uORF/CDSpairs. TheseuORFsare enriched in AUG initiation sites, non-overlapping, and shorterin length. Cumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE withneuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationshipsand potential regulatory functionsof uORF/CDS pairs in a model of neuronal differentiation. Examination of upstream open reading frame dependent translation regulation across non-differentiated and RA-differentiated SH-SY5Y cells