Monosomes actively translate synaptic mRNAs in neuronal processes

Abstract

To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.

Ribosome heterogeneity in stem cells and development

“…mRNAs with poor translation initiation rates are typically more sensitive to ribosome concentration fluctuations, and certain cell types and tissues that reply on the protein levels of such mRNAs are consequently more vulnerable to perturbations in ribosome concentration (Lodish, 1974;Mills and Green, 2017), such as the important erythroid transcription factor GATA1 in the context of Diamond-Blackfan anemia (Khajuria et al, 2018;Ludwig et al, 2014). The ribosome concentration model was further supported by a recent study demonstrating a preferential translation of synaptic mRNAs by monosomes, but not polysomes, in neuronal processes (Biever et al, 2020). For more information on the ribosome concentration model, readers are referred to the review by Mills and Green (2017).…”

Puromycin reactivity does not accurately localize translation at the subcellular level

“…While initial reports argued that emetine was required to stabilize the interaction of puromycylated peptides with ribosomes, some recent studies of local protein synthesis via the puromycylation method relied on treatment with puromycin alone for ~5-10 min, with the implication that detected nascent proteins do not appreciably diffuse away from their site of synthesis (i.e. ribosome) within the treatment time (13,14,33). To determine how far a nascent protein might diffuse on these timescales (i.e.…”

“…However, even in the most limiting case of onedimensional diffusion-approximating movement along a very narrow neural projection-a protein is expected to diffuse ~100 um in less than 1 min. This distance is large compared to both the scale of the relevant structures to which protein synthesis was localized in neurons (tens of microns) (12,13), and to the diameter of HeLa cells (~20 microns) (35), in which the method was demonstrated (8). Thus, limiting puromycin treatment time to a few minutes does not ensure that nascent proteins remain confined to the subcellular region in which they are synthesized.…”

“…Cycloheximide and emetine, in fact, are sometimes omitted from in vivo puromycylation assays based on the claim that "short" (minutes) labeling times capture peptides near their original site of translation before significant diffusion has taken place. Puromycin-based imaging methods have been widely adopted, particularly in neurobiology, where translation in neuronal processes, far from the cell body, is crucial to neuronal function (12)(13)(14)(15)(16)(17)(18)(19)(20). Other studies have combined puromycin treatment with proximity-dependent ligation (PLA) to monitor the location of translation of a specific protein (12), but this method again does not address diffusion of puromycylated peptides post-release from the ribosome.…”

Comprehensive Genome-Wide Approaches to Activity-Dependent Translational Control in Neurons

“…A purification method of the total RNAs from the same cell origin regardless of ribosome association should be developed. Recently, Biever et al combined microdissection of the neuropil (axon-enriched region) and somata (cell-body-enriched region) of the CA1 area of the hippocampus with polysome profiling and ribosome profiling, demonstrating that a specific subset of mRNAs are preferentially translated by monosomes in the neuropil, while somatic translation is mediated by polysomes [105]. As shown in their study, ribosome profiling can be exploited to uncover or explain subcellular controls of translation as well as cell-type specific ones, if the technique is combined with an appropriate isolation method.…”