Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling (Microarray). Mus musculus

Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Overall design: Untreated TSC2 WT MEFs, TSC2 KO MEFs and TSC2 WT MEFs, TSC2 KO MEFs treated with 20nM rapamycin for 30 minutes or 3hours were harvested for ribosme profiling. The fraction samples were pooled into three groups based on velocity sedimentation: single ribosome fraction (Small group), fractions with 2 ~ 4 ribosomes (Medium group), and the one with ≥5 ribosomes (Large group). RNA were extracted from the whole cell lysis and each fraction group.