Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

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.

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

“…This was shown by comparing the RSD (relative standard deviation) of the mass signal (RSD∼24%) over 12 weeks with the RSD of the response factors (RSD∼2%). Consequently, the method allows, in addition to comparison of the relative modification content of related samples, an absolute quantification, limited to those nucleosides available in weighable quantities (Buchhaupt et al, 2014; Spenkuch et al, 2014). The method also extends the linear dynamic range for quantification by two orders-of-magnitude by equalizing ion suppression effects due to saturation (gray area in Fig.…”

Absolute Quantification of Noncoding RNA by Microscale Thermophoresis

“…Most recent developments in RNAq uantification concern library preparations for RNA-Seq. [5] Thei nflection point, corresponding to 50 %o fb ound ligand, is ah alf-maximum value, [1] Concerning noncoding RNAs,s everal recent reports particularly focused on tRNAq uantification.…”

“…Considering that the K D values are in the pm range for a1 6mer-cDNA, [3] af ull-length cDNAc an be safely assumed to quantitatively hybridize to its complementary tRNAa tl east in the nm range.Intitration experiments,constant amounts of fluorescently labeled cDNAp robes (FCPs) were hybridized with increasing amounts of target tRNA, here an in vitro transcript (IVT) of tRNA Met (CAU) from E. coli. [5] Thei nflection point, corresponding to 50 %o fb ound ligand, is ah alf-maximum value, that is,intypical applications,essentially equivalent to the K D value. MST uses fluorescence detection to monitor the directed diffusion of biomolecules along at emperature gradient for the quantitative analysis of bimolecular binding events.…”

Absolute Quantifizierung nicht‐kodierender RNA‐Spezies mittels Mikroskala‐Thermophorese

“…Unter Berücksichtigung von K D -Werten im pM-Bereich bei einer 16mer-cDNA [3] kann davon ausgegangen werden, dass eine cDNAinvoller tRNALänge quantitativ an ihre komplementäre tRNA, zumindest bis in den nm-Bereich, hybridisiert. [5] Der Wendepunkt, der 50 %g ebundenen Liganden wiederspiegelt, entspricht als Halbmaximalwert in typischen Anwendungen im Wesentlichen dem K D . Um die Menge an tRNA-cDNA-Duplex zu bestimmen, haben wir sowohl einen Electrophoretic-Mobility-Shift-Assay (EMSA) mittels nicht-denaturierender PAGE als auch die Mikroskala-Thermophorese (MST) getestet.…”

“…Eine typische Darstellung der Komplex-vs.Ligand-Konzentration ergibt bei logarithmischer Skalierung eine klassische sigmoidale Kurve. [5] Der Wendepunkt, der 50 %g ebundenen Liganden wiederspiegelt, entspricht als Halbmaximalwert in typischen Anwendungen im Wesentlichen dem K D .…”