Thermodynamic contribution and nearest-neighbor parameters of pseudouridine-adenosine base pairs in oligoribonucleotides

Abstract

Pseudouridine (Ψ) is the most common noncanonical nucleotide present in naturally occurring RNA and serves a variety of roles in the cell, typically appearing where structural stability is crucial to function. Ψ residues are isomerized from native uridine residues by a class of highly conserved enzymes known as pseudouridine synthases. In order to quantify the thermodynamic impact of pseudouridylation on U-A base pairs, 24 oligoribonucleotides, 16 internal and eight terminal Ψ-A oligoribonucleotides, were thermodynamically characterized via optical melting experiments. The thermodynamic parameters derived from two-state fits were used to generate linearly independent parameters for use in secondary structure prediction algorithms using the nearestneighbor model. On average, internally pseudouridylated duplexes were 1.7 kcal/mol more stable than their U-A counterparts, and terminally pseudouridylated duplexes were 1.0 kcal/mol more stable than their U-A equivalents. Due to the fact that Ψ-A pairs maintain the same Watson-Crick hydrogen bonding capabilities as the parent U-A pair in A-form RNA, the difference in stability due to pseudouridylation was attributed to two possible sources: the novel hydrogen bonding capabilities of the newly relocated imino group as well as the novel stacking interactions afforded by the electronic configuration of the Ψ residue. The newly derived nearest-neighbor parameters for Ψ-A base pairs may be used in conjunction with other nearest-neighbor parameters for accurately predicting the most likely secondary structure of A-form RNA containing Ψ-A base pairs.

Nearest-neighbor parameters for 7-deaza-adenosine·uridine base pairs in RNA duplexes

“…The average deviations between the predicted and experimental values were 3.2%, 2.7%, and 3.2% for ΔG°3 7 , ΔH°, and ΔS°, respectively. These values are comparable to those previously reported for standard Watson-Crick pairs (3.2%, 6.0%, 6.8%, respectively) (Xia et al 1998), inosine-uridine base pairs (5.1%, 4.6%, 5.1%, respectively) (Wright et al 2007), and pseudouridine-adenosine base pairs (1.7%, 6.7%, 8.0%, respectively) (Hudson et al 2013). …”

“…Recent studies with RNA duplexes containing the nonstandard nucleotides inosine (I) and pseudouridine (Ψ) have reported noteworthy changes in RNA thermodynamic stability after conversion from the corresponding standard nucleotides (Wright et al 2007;Hudson et al 2013). When an adenosine (A) is converted to an I as a result of oxidative deamination by an enzyme family known as adenosine deaminases that act on RNA (ADARs), numerous changes take place (Fig.…”

“…In this instance, the number and types of atoms remain the same and no base shifting is required to hydrogen bond with A; however, a rearrangement occurs in which the nitrogen on the sugar face is moved to the Hoogsteen face, and the double bond is moved within the ring, which likely alters the stacking interactions of this base pair with the neighboring base pairs. Hudson et al (2013) reported that a duplex containing an internal Ψ·A pair is, on average, 1.7 kcal/mol more stable than the duplex with the corresponding U-A pair. With the recent insight that the modifications I and Ψ yield significant changes in stability, it would be beneficial to better understand how small changes to standard base pairs change the stability of RNA duplexes.…”

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

“…In both human and Toxoplasma mRNAs, Ψ is disproportionately present at position 1 of the codon, and relatively absent at position 3. This is particularly intriguing in light of the structural and stabilizing role that Ψ can play in base interactions (Hudson et al 2013;Kierzek et al 2014). Because a Ψ-A base pair may be stronger than a U-A base pair, modification of position 1 of the codon could increase the fidelity of translation.…”

“…The role of Ψ remains poorly understood, although it is known that Ψ can affect the secondary structure and basepairing of RNA (Arnez and Steitz 1994;Davis 1995;Newby and Greenbaum 2002a;Hudson et al 2013;Kierzek et al 2014). In this way, Ψ's can stabilize tRNA folding and increase the fidelity of codon-anticodon interactions, stabilize certain stem-loop conformations of snRNAs, and enable ribosome assembly (Yarian et al 1999;Greenbaum 2001, 2002b;Nobles et al 2002;Cabello-Villegas and Nikonowicz 2005;Denmon et al 2011;Jack et al 2011;Penzo et al 2015).…”