Ribo-ITP coupled ribosome profiling identifies non-canonical translational events in low input samples
In the last decade,  an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we employed Ribo-ITP, a low input ribosome profiling technique on difficult to collect samples like microdissected hippocampal tissues and single pre-implantation embryos. We detected thousands of translons across these samples, most of which initiated at non-canonical start sites, were less than 100 amino long with significantly altered amino acid composition. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed pervasive expression of most of the detected translons. To test the translational capacity of the detected translons, we engineered a translon dependent GFP reporter system, and detected robust expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from ultra-low input samples which can be applied to cell and tissue types inaccessible to conventional methods. Overall design: To identify non-canonical translation events, ribosome profiling from low input samples - microdissected mouse hippocampal acute slices and mouse preimplantation embryos was performed followed by pooled Crispr-Cas9 screen to calidate the identified events.