Explore the vast range of titles available.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2Δ) revealed global translation defects during recovery from stationary phase with translation of most genes reduced more than 4-fold. Ribosomes accumulated at start codons, were depleted from stop codons, and showed codon-specific changes in occupancy in lso2Δ. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology.

Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. Ribosomes are structures within cells that are responsible for making proteins. Molecules called messenger RNAs (or mRNAs), which contain genetic information derived from the DNA of a gene, pass through ribosomes that then “translate” that information to build proteins. Although all living cells contain ribosomes, the protein building blocks that make up the structure of the ribosome are not the same in all species. Furthermore, the exact roles that each building block plays during translation are not known. The ribosomes of plants, animals, and budding yeast contain the same protein, known as Asc1 in budding yeast and RACK1 in plants and animals. Thompson et al. have now explored the role of Asc1 in yeast cells by measuring translation in the absence of Asc1 using a technique called ribosome footprint profiling. This analysis revealed that cells lacking Asc1 translate fewer short mRNA molecules than normal cells. Short mRNAs encode small proteins that tend to play important ‘housekeeping’ roles in the cell — by forming the structural building blocks of ribosomes, for example. It has been observed previously that short mRNAs are translated at a higher rate than longer mRNAs on average, although the reasons behind this bias are still mysterious. The findings of Thompson et al. suggest that the ribosome itself may discriminate between short and long mRNAs and that the Asc1 protein is involved in calibrating the ribosome’s preference for short mRNAs. Cells need differing amounts of small proteins in different growth conditions. It will therefore be interesting to investigate whether mRNA length discrimination can be regulated by Asc1 and/or other components of the ribosome to tune gene expression to the environment.

The modification of uridine to pseudouridine is widespread in transfer and ribosomal RNAs but not observed so far in a coding RNA; here a new technique is used to detect this modification on a genome-wide scale, leading to the identification of pseudouridylation in messenger RNAs as well as almost 100 new sites in non-coding RNAs. Naturally pseudouridylated mRNAs The modification of uridine to pseudouridine is widespread in transfer and ribosomal RNAs. Until now it had not been identified in a coding RNA. Wendy Gilbert and colleagues have employed a next-generation sequencing technique called Pseudo-seq to measure the uridine-to-pseudouridine modification on a genome-wide scale. They not only identify almost 100 new sites in non-coding RNAs, but also discover pseudouridylation in mRNAs. These modifications are mostly due to the activity of the Pus pseudouridine synthase family members, and they can be responsive to environmental conditions, suggesting that inducible modification of RNA might be another way to regulate transcription. Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs 1 , enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure 2 , 3 , 4 , 5 , 6 , 7 , 8 . Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre 9 , 10 . However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease 11 , 12 , 13 .

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease. This study presents dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of this RNA modification with single-nucleotide resolution, revealing dihydrouridine at new locations across the yeast transcriptome that suggest a broad role in folding functional RNA structures.

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5′ untranslated regions that confer thousand-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we identified novel translational enhancers and silencers, determined a functional role for most alternative 5′ UTR isoforms expressed in yeast, and revealed a general mode of increased translation via direct binding to a core translation factor. DART enables systematic assessment of the translational regulatory potential of 5′ UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems. DART illuminates thousand-fold differences in 5′ UTR-specific translation activity SNPs and alternative 5′ UTR isoforms affect ribosome recruitment significantly Inhibitory effects of RNA structures are highly dependent on 5′ UTR context 5′ UTR motifs bind initiation factors directly, broadly stimulating translation

This study investigates the effects of tropospheric ozone (O3), a potent greenhouse gas and air pollutant, on European forests, an issue lacking comprehensive analysis at the site level. Unlike other greenhouse gases, tropospheric O3 is primarily formed through photochemical reactions, and it significantly impairs vegetation productivity and carbon fixation, thereby affecting forest health and ecosystem services. We utilise data from multiple European flux tower sites and integrate statistical and mechanistic modelling approaches to simulate O3 impacts on photosynthesis and stomatal conductance. The study examines six key forest sites across Europe – Hyytiälä and Värriö (Finland), Brasschaat (Belgium), Fontainebleau-Barbeau (France), and Bosco-Fontana and Castelporziano 2 (Italy) – representing boreal, temperate and Mediterranean climates. These sites provide a diverse range of environmental conditions and forest types, enabling a comprehensive assessment of O3 effects on gross primary production (GPP). We calibrated the Joint UK Land Environment Simulator (JULES) model using observed GPP data to simulate different O3 exposure sensitivities. Incorporating O3 effects improved the model's accuracy across all sites, although the magnitude of improvement varied depending on site-specific factors such as vegetation type, climate and ozone exposure levels. The GPP reduction due to ozone exposure varied considerably across sites, with annual mean reductions ranging from 1.04 % at Värriö to 6.2 % at Bosco-Fontana. These findings emphasise the need to account for local environmental conditions when assessing ozone stress on forests. This study highlights key model strengths and limitations in representing O3–vegetation interactions, with implications for improving forest productivity simulations under future air pollution scenarios. The model effectively captures the diurnal and seasonal variability of GPP and its sensitivity to O3 stress, particularly in boreal and temperate forests. However, its performance is limited in Mediterranean ecosystems, where pronounced O3 peaks and environmental stressors such as high vapour pressure deficit exacerbate GPP declines, pointing to the need for improved parameterisation and representation of site-specific processes. By integrating in situ measurements, this research contributes to developing targeted strategies for mitigating the adverse effects of O3 on forest ecosystems.