Accurate termination of protein synthesis is paramount for the integrity of cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding validated by massively paralleled reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogenous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing arises from post-decoding mRNA scanning by the 3’ end of 18S rRNA. We further uncover tissue-specific patterns of termination pausing that correlates with the stoichiometry of Rps26, which modulates mRNA:rRNA interactions. Together, these results establish termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.
Accurate termination of protein synthesis is paramount for the integrity of the cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding supported by massively parallel reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogeneous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing is consistent with post-decoding mRNA scanning by the 3′ end of 18 S rRNA. We further uncover tissue-specific patterns of termination pausing that correlate with the stoichiometry of Rps26, which potentially modulates mRNA:rRNA interactions. Together, these results suggest termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.