Abstract
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.
Highlights
The canon of known nucleoside building blocks that occur naturally in nucleic acids has rapidly expanded in the last two decades [1,2,3]
We chose yeast (Saccharomyces cerevisiae) tRNAPhe, a gold standard in the field, as substrate, whose structure and function have been characterized in abundance [53,54]
Analysis by SDS-PAGE showed an SDS-stable complex in ∼40% yield, which was specific for 5FU55 (Figure 2) and could be disrupted by heating to 95◦C in SDS loading dye. This complex essentially reproduces a previous report using a 5FU-containing T stem-loop (TSL) minimal substrate, interpreted as a covalent complex resulting from suicide action of 5FU [23]
Summary
The canon of known nucleoside building blocks that occur naturally in nucleic acids has rapidly expanded in the last two decades [1,2,3] Nucleoside modifications owe their recent surge in popularity to their involvement in epigenetic phenomena, both DNA and RNA related, which have been uncovered by newly emerging technologies, in particular by deep sequencing approaches [4]. There is a renewed interest in modification enzymes and their interaction with nucleic acids This applies to the interaction of RNA and pseudouridine synthases (Pus) [5,6], which have been investigated for decades, key features, such as, e.g. catalytic mechanism [7,8], remain elusive. Most striking among the many functions and locations [9,10,11,12,13] of this most abundant RNA modification [14] is the recent observation of its capability to alter message decoding [15,16] and the recent discovery of its widespread distribution in eukaryotic mRNAs [17,18], putting it square in the bulls eye of epigenetic research along with, e.g. m6A [19,20]
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