Sequence-intrinsic barriers define a class of translation-restricted uORFs in the human genome

Ribosome engineering enhances genetic code expansion in Saccharomyces cerevisiae

Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.

Large-scale structural analysis of pre-mRNA structure using RADIS

16S rRNA Modifications Are Dispensable for Viability but Collectively Optimize Ribosome Biogenesis and Translation Initiation in Escherichia coli

Ribosomal RNAs contain numerous conserved nucleotide modifications, yet the functional importance of most of these modifications remains unclear. In Escherichia coli, deletion of individual 16S rRNA modification enzymes generally produces only minor phenotypes, raising questions about their biological significance. Here, we generated a comprehensive collection of deletion mutants lacking individual and combined 16S rRNA modifications, culminating in a strain lacking all known 30S ribosomal subunit modifications. Despite the absence of all known 16S rRNA modifications, cells remained viable, exhibiting a fitness defect of ∼30% at 37 °C that increased to ∼50% at 20°C, consistent with impaired ribosome biogenesis. We identified strong epistatic interactions between modifications in the 3ʹ major and 3ʹ minor domains of 16S rRNA, resulting in disproportionately large effects on both fitness and antibiotic susceptibility. Live-cell single-molecule tracking revealed a marked increase in the fraction of non-translating ribosomes and a prolonged time required to enter productive translation, whereas translational elongation by actively engaged 70S ribosomes remained largely unaffected. In addition, fluorescence-based measurements showed that unmodified ribosomes exhibited increased stringency during translation initiation, reducing utilization of near-cognate start codons. Together, these findings demonstrate that 16S rRNA modifications are not essential for viability but collectively enhance the efficiency, robustness, and fidelity of ribosome assembly and translational initiation.

The conserved β-hairpin of the SUI1 domain is a dual-function structural module governing translation initiation and ribosome recycling in yeast

Most eukaryotic mRNAs encode a single functional polypeptide. Following translation termination, both the large and small ribosomal subunits are typically released from the mRNA by ribosome recycling factors. However, after translating short upstream open reading frames (uORFs) within the 5’ untranslated regions (UTRs), ribosomes can remain associated with the mRNA and reinitiate translation. This process is regulated by the heterodimer MCTS1•DENR (Tma20p•Tma22p in yeast). DENR/Tma22p harbors a SUI1 domain, structurally homologous to the translation initiation factor eIF1/Sui1p, which features a conserved, positively charged β-hairpin loop critical for eIF1 function. Despite this structural similarity, the functional significance of specific elements within DENR/Tma22p remains unexplored. Here, we used in vivo reporter assays in Saccharomyces cerevisiae to quantify reinitiation efficiency following translation of either a short uORF (in the 5’ UTR) or a full-length coding sequence (in the 3’ UTR). Systematic analysis of single, double, and triple deletions of TMA20, TMA22, and TMA64 (a homolog of Tma20p•Tma22p) revealed that the Tma20p•Tma22p complex exerts a dominant role over Tma64p in modulating reinitiation, while exhibiting functional interplay between the two factors. Using knockout strains complemented with Tma22p variants, we further demonstrated that the positively charged residues of the β-hairpin loop 1 are essential for Tma22p recycling activity. Unexpectedly, deletion of the entire SUI1 domain was less deleterious, and eIF1/Sui1p was able to partially substitute for the SUI1 domain of Tma22p within a chimeric protein context. Our findings establish the β-hairpin loop 1 of the DENR/Tma22p SUI1 domain as a critical determinant for ribosome recycling and reinitiation, and raise the question of whether MCTS1/Tma20p can promiscuously operate with either DENR/Tma22p or eIF1/Sui1p – two specialized factors that evolved from a common structural scaffold to govern distinct steps in the translation cycle.

Functional atlas of vertebrate viral RNA elements that stabilize RNA and enhance translation.

Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.

Structure-guided antisense oligonucleotides selectively modulate frameshifting of a human gene

Programmed -1 ribosomal frameshifting (-1 PRF) is a conserved translational recoding mechanism that expands proteomic diversity and regulates gene expression through RNA structural elements, most notably stimulatory pseudoknots. This mechanism is common in viruses, where it is used to control stoichiometry of viral protein products generated by the host cell to direct viral replication. Despite its biological importance, strategies to selectively modulate frameshifting remain limited. The mammalian retrotransposon-derived gene PEG10 also relies on -1 PRF to produce a fusion protein, gag-pol, which is necessary for reproduction but has also been implicated in neurological diseases. Here, we establish an antisense oligonucleotide (ASO) targeting an RNA structural element as an effective approach to tune PEG10 frameshifting. Using structure prediction, systematic antisense tiling across the PEG10 pseudoknot, and multiple model systems, we identify a discrete functional hotspot within the lower RNA stem that governs frameshift efficiency. ASOs targeting this region selectively suppress gag-pol production with minimal impact on gag, thereby shifting the ratio of protein products in a dose-dependent manner. Mechanistic dissection using RNase H-active and -inactive ASO designs, pre-annealed duplexes, and fluorescence-based subcellular localization supports a predominantly nuclear mode of action in which ASOs engage nascent PEG10 transcripts and bias pseudoknot folding away from the frameshift-competent conformation. Functional effects are conserved between human cell lines and murine models, including neurons, highlighting the generality of this strategy. Together, our results define RNA structural dynamics as a druggable layer of translational regulation and establish antisense modulation of pseudoknot folding as a way to control endogenous frameshifting. This work provides a conceptual and practical framework for targeting recoding-dependent gene products such as PEG10 in disease and suggests broader applicability of structure-directed ASOs to viral and cellular frameshifting elements.