Monitoring translation in synaptic fractions using a ribosome profiling strategy

Abstract

Background: The aim of this study was to develop a method to study genome-wide local translation in biochemically isolated synaptic fractions (synaptoneurosomes). This methodology is of particular interest for neurons, due to the cardinal role of local translational control in neuronal sub-compartments, such as dendrites, for plasticity, learning, memory, and for disorders of the nervous system.

New method: We combined established methods for purifying synaptoneurosomes with translational profiling (ribosome profiling), a method that employs unbiased next generation sequencing to simultaneously assess transcription and translation in a single sample.

Results: The two existing methods are compatible to use in combination and yield high quality sequencing data, which are specific to synaptic compartments. This new protocol provides an easy to implement workflow, which combines biochemical isolation of synaptoneurosomes of varying levels of purity (crude or Percoll gradient purified) with the use of a commercial kit to generate sequencing libraries.

Comparison with existing methods: Compared to previous studies of the synaptic translatome, our method shows less contamination with non-neuronal cell types or non-synaptic compartments, increasing the specificity of the data obtained.

Conclusions: Combining the isolation of functional synaptic units with ribosome profiling offers a powerful tool to study local translation in synaptic compartments both in health and disease.

Keywords: Local translation; Ribosome profiling; Synapse; Synaptoneurosomes; mRNA translation.