GSE99920
Transcriptional and ribosome profiling of Drosophila 3rd instar larvae muscle
Presynaptic efficacy can be modulated by retrograde control mechanisms, but the nature of these complex signaling systems remain obscure. Some studies have suggested that retrograde synaptic communication requires new protein synthesis in the postsynaptic compartment, yet the putative translational targets that mediate this signaling are enigmatic. To gain insight into the retrograde mechanisms that stabilize synaptic transmission at the neuromuscular junction, we have developed and optimized a tissue-specific ribosome profiling approach in Drosophila. We first demonstrate the ability of this technology to define genome-wide translational regulations that could not be inferred from transcription, and validate the superior sensitivity of ribosome profiling over conventional translational profiling. We then leverage this technology to test the relative contributions of transcriptional, translational, and post-translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function. Surprisingly, we find no changes in transcription or translation are necessary to enable retrograde homeostatic signaling. Rather, post-translational mechanisms appear to ultimately gate instructive retrograde communication. Finally, we find that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this demonstrates the power of ribosome profiling to define transcriptional, translational, and post-translational mechanisms driving retrograde signaling during adaptive plasticity. Overall design: Transcriptional (RNA-seq of total RNA) and ribosome profiling were performed in Drosophila muscle tissue. Three genotypes were analyzed: wild type (control sample), GluRIIA mutant and Tor muscle overexpression. In addition, translating ribosome affinity purification (TRAP) based translational profiling was done in wild type sample to assay the difference between ribosome profiling and TRAP translational profiling. Three replicates were performed for each analysis.