Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain.

Neuronal protein synthesis is highly compartmentalised and regulated, with key roles for translation initiation and elongation factors. Ribosome profiling, the most widely used transcriptome-wide method for measuring translation, captures translation elongation, but not the initiation phase involving small ribosomal subunit (SSU) scanning. Here, we adapted ribosome complex profiling (RCP-seq) for mouse dentate gyrus and cerebral cortex, to characterize translation initiation. In both tissues, SSUs accumulate near the start codon on synaptically localised RNAs, and this 'poised' SSU configuration correlates with enhanced translational efficiency. Upstream open reading frames (uORFs) are associated with less poised SSUs, potentially by disassociating the SSUs. We further find that neuron-specific transcripts recruit more ribosomes and are more efficiently translated than glia-specific transcripts. For neuronal transcripts, monosome-preferring mRNAs show less SSU occupancy relative to polysome-preferring mRNAs, suggesting reduced recruitment of ribosomes. In summary, RCP-seq elucidates translation initiation dynamics and cell-type- and transcript-specific regulation in the mammalian brain.