Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. 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. 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.American Cancer Society (Robbie Sue Mudd Kidney Cancer Research Scholar Grant RSG-13-396-01-RMC)National Institutes of Health (U.S.) (GM094303)National Institutes of Health (U.S.) (GM081399)American Cancer Society. New England Division (Ellison Foundation Postdoctoral Fellowship)American Cancer Society (Postdoctoral Fellowship PF-13-319-01-RMC)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287

tRNA structural and functional changes induced by oxidative stress

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level

The poly-omics of ageing through individual-based metabolic modelling

Abstract Background Ageing can be classified in two different ways, chronological ageing and biological ageing. While chronological age is a measure of the time that has passed since birth, biological (also known as transcriptomic) ageing is defined by how time and the environment affect an individual in comparison to other individuals of the same chronological age. Recent research studies have shown that transcriptomic age is associated with certain genes, and that each of those genes has an effect size. Using these effect sizes we can calculate the transcriptomic age of an individual from their age-associated gene expression levels. The limitation of this approach is that it does not consider how these changes in gene expression affect the metabolism of individuals and hence their observable cellular phenotype. Results We propose a method based on poly-omic constraint-based models and machine learning in order to further the understanding of transcriptomic ageing. We use normalised CD4 T-cell gene expression data from peripheral blood mononuclear cells in 499 healthy individuals to create individual metabolic models. These models are then combined with a transcriptomic age predictor and chronological age to provide new insights into the differences between transcriptomic and chronological ageing. As a result, we propose a novel metabolic age predictor. Conclusions We show that our poly-omic predictors provide a more detailed analysis of transcriptomic ageing compared to gene-based approaches, and represent a basis for furthering our knowledge of the ageing mechanisms in human cells

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.</p