Mapping the small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain
Protein synthesis in neurons is highly compartmentalized and regulated, with key roles for translation initiation and elongation factors. Transcriptome-wide methods for measuring translation, such as ribosome profiling, assess the elongation phase of translation but are blind to the initiation phase with scanning of the small ribosomal subunit (SSU). Here, we optimized a UV fixation method for ribosome complex profiling (RCP-seq), allowing analysis of translation initiation dynamics in the adult murine cerebral cortex and dentate gyrus brain regions. We demonstrate region- and gene-specific differences during scanning and elongation and preferential translation of 5' upstream open reading frames (uORFs) in the dentate gyrus vs the cortex. We showed that neuron-specific transcripts are more expressed, scanned and translated relative to glia-specific transcripts and that scanning is reduced in monosome-preferring neuronal RNAs relative to those that prefer polysome-translation.  Finally, we showed an extensive accumulation of SSUs upstream of the start codon on synaptically localized RNAs. In sum, RCP-seq elucidates translation initiation dynamics in the mammalian brain and uncovers brain region-, cell-type-, and transcript-specific regulation.