Pseudouridine (C) is the most abundant of >150 nucleoside modifications in RNA. Although C was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of C synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC–MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations <2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude.

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.

Here we investigated the stability of an aptamer, which is formed by two RNA-strands and binds the antibiotic streptomycin. Molecular dynamics simulations in aqueous solution confirmed the geometry and the pattern of hydrogen bond interactions that was derived from the crystalstructure (1NTB). The result of umbrella sampling simulations indicated a favored streptomycinbinding with a free energy of ‫ܩ∆‬ ୠ୧୬ୢ°= -101.7 kJ mol -1 . Experimentally, the increase in oligonucleotide stability upon binding of streptomycin was probed by single-molecule force spectroscopy. Rate dependent force spectroscopy measurements revealed a decrease in the natural off-rate (k off-COMPLEX = 0.22 ± 0.16 s -1 ) for the aptamer-streptomycin complex compared to the aptamer having an empty binding pocket (k off-APTAMER = 0.49 ± 0.11 s -1 ). This decrease in the natural off-rate corresponds to a decrease in the Gibbs free energy of ∆∆G ୱ୦ୣୣ୰ ≈ 3.4 kJ mol -1 .The simulated binding pattern and the experimental results led to the conclusion that hydrogen-bonds between both RNA strands mainly contribute to the decrease in natural-off rate and Gibbs free energy of the aptamer system studied.

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