Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5 cap. Consequently, only one ribosome scans a 5UTR at a time, and 5UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of [~]12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.\n\nHIGHLIGHTSO_LISelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivo\nC_LIO_LIScanning ribosomes are cap-tethered in human cells\nC_LIO_LIOnly one ribosome scans a 5UTR at a time in human cells\nC_LIO_LIRibosomes retain eIFs during early translation, allowing reinitiation after uORFs\nC_LI