Ribosome profiling in RPEI cells upon CDK1-inhibition

Protein synthesis is the second most energy-intensive process in the cell. Therefore, cells tightly control mRNA translation rates in response to external stimuli (growth factors, differentiation signals) and the internal resources available (nutrients, energy). We set out to unravel new molecular mechanisms of global translation control required for cellular homeostasis. Using a siRNA screening approach to knock-down all human kinases and phosphatases, we analyzed the impact of protein phosphorylation on translation regulation in unstressed, proliferating cells. A visual screen was conducted to assess stress granule formation (as a consequence of translational shutdown) in the absence of 711 kinases and 256 phosphatases. Several candidates identified in the screen are linked, either directly or indirectly, to cell cycle regulation. We further identified a cyclin-dependent kinase (CDK), whose genetic downregulation or chemical inhibition results in global translation suppression. In order to assess CDK downstream signaling, we performed phosphoproteomics analysis and found the phosphorylation status of many ribosomal proteins to be altered upon CDK inhibition. Indeed, we could show that S6-kinase 1 partially contributes to CDK-dependent translational control. Ribosome profiling further revealed that the translation of 5'TOP mRNAs, including many mRNAs encoding for ribosomal proteins, is especially sensitive to CDK inhibition. While the translation regulatory function of this kinase is independent of the cell cycle phase, its activity is ideally suited to couple the overall proliferation rate of cells to the global rate of protein synthesis. Our results identify a pathway that enables cells to directly adapt protein homeostasis to proliferation and growth signals. Overall design: RO-3306 or DMSO treated RPEI cells were subjected to ribosome profiling in three biological replicates. As a spike-in to detect global changes in translation efficiency, S. cerevisiae lysate was added to the RPEI lysates. To determine ribosome densities, both ribosome protected fragments and alkaline-fragmented input material were analyzed, amounting to 12 samples in total.