Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Researchers at the Columbia University Irving Medical Center have now developed riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). The researchers compared RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.
Overviews of the protocol and experimental design of the study performed
(a) Schematic diagram of the riboPLATE-seq protocol, from lysis in a multi-well plate to pooled library preparation. The right-hand side mirrors the original PLATE-seq protocol. In this workflow, an oligo(dT)-grafted plate captures polyadenylated RNA that can be reverse-transcribed with barcoded adapters, generating a plate of cDNA that may be pooled for library construction. The left side incorporates a pan-ribosome IP before PLATE-seq pooling and library preparation, generating instead a pooled library of ribosome-associated RNA. (b) Simplified structure of the signaling pathways under study and the specific protein targets considered. The PI3K/AKT/mTOR signaling axis at left converges with the MAPK/ERK pathway at right on eIF4E, early in the process of ribosome assembly (green box). The figure also outlines the inhibitors used in this study and their specific targets within these pathways. NVP-BKM120 is a PI3K inhibitor (orange), both AZD8055 and PP242 are mTOR inhibitors (blue and purple, respectively), MNK-i1 is a MNK1 inhibitor (green), and 4EGi-1 is a direct eIF4E inhibitor (black).