From synaptic plasticity including memory functions, to adaptation to immediate environments and stresses, eukaryotic cells rely on fast-paced translational control. Many protein factors involved in translation are known oncogenes, or their functions are dysregulated in malignancies. To understand how the cells mitigate adverse conditions or acquire and maintain a malignant phenotype, it is critical to expose the underlying regulation of protein biosynthesis from mRNA.
We use an array of in vivo methods based on RNA-protein crosslinking to investigate the dynamics of RNA-level cellular control. Applying translation complex profile sequencing (TCP-seq) [Archer SK et al. Nature 2016; Shirokikh NE et al. Nat. Protoc.], we detect extremely rapid (seconds to minutes) changes in translation concomitant to the glucose stress response in yeast, and rapid (minutes) responses of mammalian cells to anti-cancer drugs targeting initiation. Contrasting to the global translational repression often associated with stress, we find that glucose starvation responses occur in a transcript-specific manner, with gluconeogenic mRNAs substantially upregulated. We capture differential utilisation of the main scanning ‘motor’ ATP-dependent RNA helicase eIF4A across mRNA using factor-specific small ribosomal subunit isolation and provide additional insights into the mechanistic role of eIF4A during ribosomal scanning of mRNA.
A major limitation of the current ‘ribosome profiling’ approaches is the inability to discern between the locations of translation rate-limiting ribosomal pausing, variable ribosomal presence across transcripts and RNA sequencing bias. We introduce direct detection of the ribosomal stall sites and mRNA abundance-independent evaluation of transcript load with the ribosomes, based on RNase-resistant disome footprint density. This allows us to precisely measure translational output from each transcript. We further use footprinting data to infer the dynamics of spatial arrangement and stability of mRNA. Our data expose previously inaccessible mechanistic details of protein biosynthesis and provide a resource for comprehensive modelling and discovery of RNA-level regulatory mechanisms.