Monosomes actively translate synaptic mRNAs in neuronal processes

INTRODUCTION RNA sequencing and in situ hybridization have revealed the presence of an unexpectedly high number of RNA species in neuronal dendrites and axons, and many studies have documented the local translation of proteins in these compartments. During messenger RNA (mRNA) translation, multiple ribosomes can occupy an individual mRNA (a complex called a polysome), resulting in the generation of multiple copies of the encoded protein. Polysomes, usually recognized in electron micrographs as a cluster of three or more ribosomes, have been detected in neuronal dendrites but are surprisingly infrequent given the diversity of mRNAs present in dendrites and axons. In neuronal processes, the features and mechanisms of translation have not been explored in detail, in part because of the relative inaccessibility of dendrites and axons. In this study, we investigated how a diverse set of neuronal proteins might be synthesized from a limited population of polysomes present in small synaptic volumes. RATIONALE To accommodate their complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. Yet a relative scarcity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a rather limited capacity for local protein synthesis. To visualize the capacity for local protein production in vivo, we profiled actively translating mRNAs in rodent hippocampal neuronal processes. To access neuronal compartments, we microdissected the neuropil and somata layer from the CA1 region of hippocampal slices, generating samples enriched in dendrites and/or axons versus cell bodies. Polysome profiling of microdissected regions was used to determine the association of axonal and/or dendritic and somatic transcripts, respectively, with monosomes or polysomes. Ribosome footprinting was then used to assess the translational activity of monosomes (single ribosomes) and polysomes. Bioinformatic analyses were used to determine the features of monosome-preferring transcripts as well as the families of protein groups that were encoded by monosome-preferring transcripts. We also compared the monosome-to-polysome (M/P) preference of transcripts between the somata and neuropil. To estimate the abundance of proteins encoded by monosome- and polysome-preferring transcripts, we measured protein levels in the neuropil by using mass spectrometry–based proteomics. RESULTS In the adult rodent brain, we detected substantial levels of ongoing protein synthesis in the synaptic neuropil (a region enriched in neuronal axons and dendrites) in vivo and provide direct evidence for the preferential translation of a high number of both pre and postsynaptic transcripts by monosomes. The monosomes were in the process of active polypeptide elongation in dendrites and axons. Most transcripts exhibited a similar M/P preference in both somata and neuropil, suggesting that ribosome occupancy is often an intrinsic feature of the transcript. Several transcripts exhibited a preference for monosomes or polysomes that switched depending on the compartment; these mRNAs encoded some synaptic plasticity–related proteins. Overall, neuropil transcripts exhibited a preference for monosome translation. Monosome-preferring transcripts encoded a full range of low- to high-abundance proteins in the neuropil. CONCLUSION In this study, we investigated the translational landscape in neuronal processes and identified local translation on 80S monosomes as an important source of synaptic proteins. Neuropil-localized transcripts exhibited a greater monosome preference than somatic transcripts, potentially allowing for the production of a more diverse set of proteins from a limited pool of available ribosomes at synapses. This finding thus bridges the gap between the relative paucity of visualized translational machinery in neuronal processes and actual measurements of local translation. Given the spatial limitations within dendritic spines and axonal boutons, synaptic activity could also regulate monosome translation to diversify the local proteome with spatial and temporal precision.