A profiling strategy to capture terminating ribosomes in mammalian cells is established and a substantial heterogeneity in ribosome pausing at individual stop codons is revealed, suggesting termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.
Abstract
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.
Stop-codon readthrough (RT) diversifies proteomes and is particularly prominent in neurons, suggesting its importance in nervous systems. However, the regulatory logic that specifies neuronal RT and the structural and cellular consequences of the resulting C-terminal protein extensions remain poorly understood. Here we leverage neuron-specific ribosome profiling datasets in Drosophila to construct an in vivo atlas of 163 neuronal RT transcripts. Sequence- based modeling distinguished RT from non-RT transcripts and highlighted an extended post-stop region enriched for stable predicted RNA structures. Beyond these cis-associated features, ribosome-footprint analysis revealed pronounced stop-codon pausing on RT transcripts, accompanied by upstream periodic peaks from the stop codon consistent with ribosome queuing. At the protein level, neuronal RT appended polypeptides enriched with polar residues and intrinsically disordered regions (IDRs). Finally, an in vivo dual-color reporter showed that RT of the RNA-binding protein Bru3 alters localization from the nucleus to cytoplasmic granules. Together, our results suggest that neuronal RT is associated with structured post-stop RNA regions that reshape termination dynamics and can produce IDR-rich C-terminal protein extensions with distinct subcellular localization.
Toshiharu Ichinose, K. Sakuma, Hiroto Anbo et al.· bioRxiv· 0 citations
It is proposed that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels that may be prevalent in bacteria which exhibit uncoupled transcription and translation.
Zachory M. Park, Christina R. Savage, Amanda R. Decker-Farrell et al.· Cell Reports· 0 citations
Stop codon readthrough is a noncanonical translation strategy employed by certain RNA viruses, in which a viral termination codon is either decoded by host near-cognate tRNAs or canonically recognized by the class I release factor (RF, eRF1 in eukaryotes). Ribosomal A-site competition between near-cognate tRNAs and eRF can shift decoding toward near-cognate tRNAs, thereby promoting non-canonical decoding events by transiently pausing termination and favoring readthrough. This review focuses on two viral cis-elements that modulate readthrough across four viral genera in which this decoding event has been experimentally validated: (i) primary sequences surrounding the stop codon (stop codon context), and (ii) downstream RNA structures. Effects of stop codon context have been observed more broadly in cellular genes, including nonsense suppression in bacteria, with mechanisms including inefficient RF association or tRNA interactions at adjacent sense codons. In eukaryotic systems, interactions with the ribosomal mRNA entry channel have been suggested. Diverse downstream structures, including gammaretroviral pseudoknots and specific structures in alpha- and coltiviruses, further stimulate readthrough in a location- and structure-sensitive manner. This effect has not been consistently observed in chikungunya and triatoviral structures, suggesting a strong dependence on local sequence and structural context. Compared with the larger number of cellular readthrough occurrences that can be detected at low efficiency by ribosome profiling, viral readthrough in mammalian systems is consistently high (>2%). Understanding the interplay between viral RNA elements and host translational machinery, including potential kinetic trapping at the termination codon, provides insights into this unusual elongation mechanism. These findings may have implications for antiviral strategies targeting these RNA elements.
Translating ribosomes can stall on mRNA for various reasons, including nuclease cleavage, arrest peptide sequences or ribosome collisions. In Escherichia coli, several ribosome rescue factors act to release ribosomes stalled at the 3' end of mRNA. Among these factors, ArfB can rescue stalled ribosomes without the help of other factors. ArfB consists of an N-terminal domain containing the catalytic GGQ motif, which mediates peptidyl-tRNA hydrolysis, and a C-terminal extension that functions as a sensor for recognizing stalled ribosomes. However, how these two regions coordinate to resolve ribosome stalling remains unclear. Here, using a reconstituted translation system, we found the functional importance of histidine residues in the N-terminal domain of ArfB. In particular, histidine at position 62 in E. coli ArfB was required for stop codon-independent fMet-tRNA hydrolysis, whereas its substitution did not affect affinity for the ribosome. Furthermore, directed hydroxyl radical probing revealed that H62A mutation did not significantly alter the overall positioning of either the N-terminal domain or the C-terminal extension of ArfB on the ribosome. These findings suggest that H62 contributes to ArfB function during a step following initial ribosome binding, thereby facilitating peptidyl-tRNA hydrolysis.
Daisuke Kurita, Kotone Furusawa, Takumi Kasuya et al.· RNA: A publication of the RN...· 0 citations
Out-of-frame translation events, arising from ribosomal frameshifting or noncanonical initiation, are an unavoidable feature of translation. Their consequences depend on the distribution of stop codons in alternative reading frames, which determines the permissiveness of those frames: whether out-of-frame translation terminates quickly or generates extended products. Using quantitative dual-fluorescence reporters, we show that these stop codons function as molecular checkpoints that terminate out-of-frame translation. Genome-wide analysis across 10 organisms reveals that natural coding sequences maintain dense stop codon distributions in alternative frames, with a median spacing of approximately 20 amino acids. Codon optimization, the standard method for enhancing translation, systematically depletes this safeguard. Because all three stop codons (UAA, UAG, UGA) begin with uridine, and optimal human codons exclude uridine from third positions, stop codons in the -1 reading frame become structurally impossible in codon-optimized sequences. Analysis of 120 therapeutic sequences, including FDA-approved COVID-19 messenger RNA (mRNA) vaccines, confirms widespread -1 frame stop codon depletion: out-of-frame products average 164 amino acids, sixfold longer than in natural human genes. Strategic restoration of stop codons through synonymous substitutions eliminates detectable out-of-frame products by mass spectrometry while preserving the intended protein. Although such products and immune responses have been detected in COVID-19 mRNA vaccine recipients, there is no evidence they cause clinical harm; nonetheless, our approach offers a simple way to eliminate them through informed sequence design alone, without changes to manufacturing or regulatory frameworks. Our findings establish stop codon distribution as a critical design parameter for protein-coding nucleic acid therapeutics.
Zheling Liu, Zhenguang Ying, Luoan Shen et al.· Proceedings of the National...· 0 citations
This work structurally characterize chimeric ribosomes derived from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae using cryo-electron microscopy and uncover a potential relationship between 16S ribosomal RNA (rRNA) stability and translation efficiency, providing new insights into rRNA structural malleability.
Tushar Raskar, Alan Costello, A. Badran et al.· Nucleic Acids Research· 0 citations
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