Author Correction: Quantitative RNA pseudouridine maps reveal multilayered translation control through plant rRNA, tRNA and mRNA pseudouridylation
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Chemical reactivity of RNA and its modifications with hydrazine
RNA modifications are essential for the regulation of cellular processes and have a key role in diseases such as cancer and neurological disorders. A major challenge in the analysis of RNA modification is the differentiation between isomers, including methylated nucleosides as well as uridine and pseudouridine. A solution is their differential chemical reactivity which enables isomer discrimination by mass spectrometry (MS) or sequencing. In this study, we systematically determine the chemical reactivity of hydrazine with RNA and its native modifications in an aniline-free environment. We optimize the conditions to achieve nearly full conversion of all uridines while avoiding RNA cleavage. We apply the conditions to native tRNAPhe which allows discrimination of pseudouridine and uridine by MALDI-MS. Furthermore, we determine the identity of the reaction product of hydrazine with various modified nucleosides using high resolution mass spectrometry and quantify the reaction yield in native tRNA from E. coli and human cells under various hydrazine conditions. Most modified nucleosides react quantitatively at lower hydrazine concentration while uridines do not decompose under these conditions. Thus, this study paves the way to exploit aniline-free hydrazine reactions in the detection of RNA modifications through MS and potentially even long-read RNA sequencing.
Analysis of microbial composition and sharing in low-biomass human milk samples: a comparison of DNA isolation and sequencing techniques
Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.
A capless hairpin-protected mRNA vaccine encoding the full-length Influenza A hemagglutinin protects mice against a lethal Influenza A infection
The success of mRNA vaccines in controlling the COVID 19 pandemic has confirmed the efficacy of synthetically synthesized mRNA in humans and has also provided a blueprint on how to design them in terms of molecular structure and cost. We describe a mRNA vector that, unlike linear mRNAs used in current vaccines/therapeutics, does not require a 5′ cap to function. The described mRNA vector initiates translation from an internal ribosomal entry site (IRES) and contains specially designed self-folding secondary structures (hairpins) to protect the 5′ end against degradation, dramatically improving its stability. The produced mRNA did not require any additional modifications for functionality. The 5′ hairpins completely inhibited cap-dependent translation, and all vectors containing them required an IRES to express protein. When this capless mRNA vector was constructed to express the full-length Influenza A membrane protein hemagglutinin (HA), complexed with pre-formed lipid-based nanoparticles, and then injected into mice as a vaccine, it generated high titers of anti-HA antibodies and protected mice against a lethal dose of Influenza A.
Comprehensive co-expression network reveals the fine-tuning of AsHSFA2c in balancing drought tolerance and growth in oat
Persistent activation of drought tolerance is detrimental to plant growth and development. However, the mechanism that balances plant drought tolerance and growth remains largely undetermined. Here, we constructed a comprehensive co-expression network comprising 84 transcriptome datasets associated with growth and drought tolerance in oats. Moreover, 84 functional modules and many candidate genes related to drought tolerance and growth were identified. A key candidate gene, AsHSFA2c was involved in fine-tuning the balance between drought tolerance and growth by inhibiting plant growth and positively regulating drought tolerance. Then, we determined AsDOF25 as an upstream positive regulator and AsAGO1 as the downstream target gene of AsHSFA2c. These results imply that the AsDOF25-AsHSFA2c-AsAGO1 module contributes to the balance between drought tolerance and growth in oats. Our findings and resources will facilitate the identification of key genes related to drought tolerance and further studies of the genetic basis underlying strong drought tolerance in oats.
Metabolite-driven mechanisms reveal chemical ecology of Lehmann Lovegrass (Eragrostis lehmanniana) invasion in North American semi-arid ecosystems
Invasive plants threaten global ecosystems, yet traditional analyses of functional traits cannot fully explain their dominance over co-occurring natives. Metabolomics offers insights into plant invasions, but single-technique studies often miss critical biochemical mechanisms. We employ a multimodal metabolomics approach (¹H NMR, LC MS/MS, FT-ICR-MS, and MALDI-MSI) to investigate the biochemical basis of Lehmann lovegrass (Eragrostis lehmanniana) invasion in semi-arid North America, comparing it with a co-occurring native grass, Arizona cottontop (Digitaria californica). Our analysis reveals three metabolomic traits of Lehmann lovegrass compared to Arizona cottontop: Enhanced nitrogen allocation in shoots, reduced defensive metabolites in root layers; and increased root exudate modulation under stress conditions. These traits suggest Lehmann lovegrass succeeds through adaptation to increasing aridity rather than direct competition, demonstrating adaptation to nutrient-poor environments and high phenotypic plasticity in response to increasing aridity. This integrated metabolomic approach provides new mechanistic insights into invasion ecology and plant adaptation under environmental change.
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