Abstract
Accurate termination of protein synthesis is paramount for the integrity of cellular proteome, but our understanding of the dynamics and fidelity of terminating ribosomes is far from complete. Here we establish profiling of terminating ribosomes in mammalian cells and report a wide range of ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that contributes to termination pausing, which was confirmed by massively paralleled reporter assays. Unexpectedly, lack of termination pausing increases the chance of stop codon slippage, generating proteins with mixed C-terminal extensions. We demonstrate that the sequence-dependent termination pausing is a result of post-decoding mRNA scanning by the 3' end of 18S rRNA. We further observe tissue-specific termination pausing that correlates with the stoichiometry of Rps26, which constrains mRNA:rRNA interaction. Thus, termination pausing represents a translational signature associated with mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.