Abstract
Experience-dependent changes to neural circuitry are shaped by spatially-restricted activity-dependent mRNA translation. Although the complexity of mRNA translation in neuronal cells is widely appreciated, translational profiles associated with neuronal excitation remain largely uncharacterized, and the associated regulatory mechanisms are poorly understood. Here, we employed ribosome profiling, mRNA sequencing and small RNA sequencing to profile transcriptome-wide changes in mRNA translation after whole cell depolarization of differentiated neuroblast cultures, and investigate the contribution of sequence-specific regulatory mechanisms. Immediately after depolarization, a functional partition between transcriptional and translational responses was uncovered, in which many mRNAs were subjected to significant changes in abundance or ribosomal occupancy, but not both. After an extended (2 h) post-stimulus rest phase, however, these changes became synchronized, suggesting that there are different layers of post-transcriptional regulation which are temporally separated but become coordinated over time. Globally, changes in mRNA abundance and translation were found to be associated with a number of intrinsic mRNA features, including mRNA length, GC% and secondary structures; however, the effect of these factors differed between both post-depolarization time-points. Furthermore, small RNA sequencing revealed that miRNAs and tRNA-derived small RNA fragments were subjected to peak changes in expression immediately after stimulation, during which these molecules were predominantly associated with fluctuations in mRNA abundance, consistent with known regulatory mechanisms. These data suggest that excitation-associated neuronal translation is subjected to extensive temporal coordination, with substantial contributions from a number of sequence-dependent regulatory mechanisms.
Highlights
Excitation of neurons is known to induce distinct patterns of gene expression which regulate activity-dependent fine-tuning of neuronal networks [1,2,3]
To investigate patterns of mRNA translation associated with membrane depolarization, differentiated SH-SY5Y cells were repeatedly stimulated with K+ and subjected to parallel ribosome profiling (Ribo-Seq) and mRNA sequencing
Previous studies have shown that overall translation is highly responsive to neuronal activity and experience-dependent plasticity [12,35,36]; discrete quantification of gene-level translation is necessary for a detailed mechanistic understanding of neuronal function
Summary
Excitation of neurons is known to induce distinct patterns of gene expression which regulate activity-dependent fine-tuning of neuronal networks [1,2,3]. Since these events are thought to underpin the molecular basis of phenomena, including learning, memory and cognition, an extensive body of studies have characterized transcriptional profiles associated with neuronal excitation in a variety of models [1,2,3]. While activity-dependent patterns of translation have been identified for a subset of neuronal genes [15,16,17,18,19], transcriptome-wide profiles of activity-dependent translation and associated regulatory mechanisms remain poorly characterized
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