Investigation of Overhauser effects between pseudouridine and water protons in RNA helices Meredith I. Newby* and Nancy L. Greenbaum*†‡ *Department of Chemistry and Biochemistry and †Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4390 Communicated by Michael Kasha, Florida State University, Tallahassee, FL, August 8, 2002 (received for review July 2, 2002) The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (␺), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uri- dine. When ␺ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The pres- ence of ␺ often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883–1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560–7567] have depicted water molecules bridging ␺NH1 groups and nearby phos- phate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. Fig. 1. Schematic structures of U and ␺ bases. R, ribose. The ␺ base is a uracil ␺ (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for NH1–water rotated about the 3–6 ring axis, so that is has a COC base–sugar linkage and cross-relaxation effects within two RNA helices: (i) a complemen- an additional protonated ring nitrogen. tary duplex, in which ␺ is not associated with structural change, and (ii) an RNA duplex representing the eukaryotic pre-mRNA branch-site helix from Saccharomyces cerevisiae, in which a con- in thermal stability, as compared with its unmodified analogue served ␺ extrudes the branch-site adenosine from the helix. Our (12, 36). The presence of ␺ results in extrusion of an adenosine, data implicate a water–␺NH1 hydrogen bond both in stabilizing the 2ЈOH of which is the nucleophile in the first step of splicing, the complementary helix and in favoring formation of the unique from the helix into a position that may facilitate its role in structure of the branch-site helix. splicing catalysis. Such studies emphasize the biological impor- tance of modified bases and may describe a means by which y 1950, it was known that RNA extracted from cells con- RNA-mediated chemical activity is enabled through site- specific, posttranscriptional modification. Btained a small percentage of a fifth base, thought to be ␺ 5-methylcytosine (1). Later, this fifth nucleoside, which ac- The mechanism by which stabilizes (and, in the case of the Ϸ pre-mRNA branch site, modifies) local RNA architecture re- counted for 4% of nucleotides in yeast transfer (t)RNA, was ␺ identified as 5-ribosyluracil, an isomer of uridine (U) (2–5). mains unclear. Stabilization of RNA secondary structure by Pseudouridine (abbreviated ␺) since has been found to be the has been hypothesized to result from additional hydrogen bonds involving the ␺NH1 (13) or from more favorable stacking most abundant modified base in tRNA, ribosomal RNA, and ␺ small nuclear (sn)RNA. Modification of U to ␺, catalyzed by a interactions between and neighboring bases (14). Corroborat- site-specific pseudouridylase, involves scission of the nucleo- ing the first hypothesis is the observation of water-mediated side’s glycosidic bond, followed by rotation of the base about its hydrogen bonds between the NH1 proton and phosphate oxygen 3–6 axis, and reattachment through the carbon at the 5-position atoms in crystal structures of tRNA molecules (ref. 15; Fig. 2) of the ring to form the only natural nucleoside with a COC and molecular dynamics simulations (8). Water-mediated hy- drogen bonds involving ␺NH1 have been postulated to stabilize base–sugar bond. The resulting modified base, which can form ␺ a Watson–Crick base pair with adenine, features an additional -containing helices in solution (13, 16), but no direct evidence ring nitrogen atom that is protonated at physiological pH (NH1; for their existence has been obtained. Experiments supporting Fig. 1; reviewed in ref. 6). the stacking hypothesis showed that 5-methyl U, which cannot The presence of this modified base has been associated with form hydrogen bonds in the major groove, at the base of a stem loop was as stable as the native ␺ modification (14). In contrast an increase in thermal stability of secondary structure (7) and ␺ has been shown to decrease the motion of neighboring bases in with all these studies, incorporation of into single-stranded molecular dynamics simulations (8), which may explain its regions and loops, in some cases, can be destabilizing (17). prevalence in structural RNAs. For example, ␺ are clustered We have sought to understand the molecular basis for stabi- lization by ␺ in a complementary RNA duplex and a duplex within the peptidyl transferase center of the ribosome (9), are BIOCHEMISTRY conserved within regions of snRNAs that are involved in RNA– representing the eukaryotic pre-mRNA branch-site helix. We RNA interactions (10), and have been implicated in spliceosome have accomplished this by use of an NMR experiment designed assembly (11). ␺ Although the presence of generally is not associated with Abbreviations: snRNA, small nuclear RNA; ROESY, rotating-frame Overhauser effect major structural change (reviewed in ref. 6), we recently have spectroscopy; NOE, nuclear Overhauser effect; ␺, pseudouridine; CLEANEX-PM, CLEAN found a major exception in the eukaryotic splicing apparatus: a chemical-exchange spectroscopy. highly conserved ␺ in the region of U2 snRNA that pairs with Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, the intron to form the precursor (pre)-mRNA branch-site helix www.rcsb.org (PDB ID codes 1LPW and 1LMV). induces a dramatic change in conformation and modest increase ‡To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.202477199 PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 12697–12702 Downloaded by guest on September 27, 2021 Fig. 2. Schematic view of a water-mediated hydrogen-bonding interaction between ␺ and water. Oxygen atoms are colored red. Here, the water mole- cule is bridging an interaction between the ␺NH1 group and phosphate Fig. 4. RNA duplexes used for investigation of water–␺ interactions. The oxygen atoms belonging to its own and the 5Ј nucleotide’s phosphate complementary duplex (␺9 ϩ 9) is shown in A, and the branch-site construct group (6). (␺9 ϩ 10) is shown in B. to identify RNA protons involved in chemical exchange with Oligomers then were resuspended in diethyl pyrocarbonate- water molecules whose transient association with the RNA is treated water, and strand concentration was measured from sufficiently long to effect cross-relaxation. This experiment, absorbance at 260 nm. Equimolar amounts of strands were called CLEANEX-PM (pulse sequence shown in Fig. 3; ref. 18), combined to obtain duplexes of the 9-bp complementary mol- previously has been used to examine interactions between ecule (␺9 ϩ 9) or the model branch site (␺9 ϩ 10), respectively. proteins and surface hydration water molecules. Despite a large Duplexes were lyophilized to dryness and resuspended in 250 ␮l difference in the structural, and perhaps functional, role of ␺ in of NMR buffer consisting of 10 mM sodium phosphate, pH 6.4, each of these contexts, we demonstrate the presence of a 50 mM sodium chloride, and 0.1 mM EDTA in 90% H2O/10% 2 cross-relaxation-induced nuclear Overhauser effect (NOE) be- H2O (99.96%; Cambridge Isotope Laboratories, Cambridge, tween the ␺NH1 proton and water protons in each duplex. This MA). Final concentration of RNA duplexes in NMR samples finding supports the model of a water-mediated hydrogen bond was Ϸ1 mM. Microvolume NMR tubes (Shigemi, Allison Park, involving ␺ as the source of added stabilization or structural PA) were used for all NMR data collection. change. NMR Experiments. A 500-MHz Varian Inova spectrometer (De- Materials and Methods partment of Chemistry and Biochemistry, Florida State Univer- Design and Synthesis of Molecular Constructs. Two RNA duplexes sity, Tallahassee) was used for data collection. Spectra acquired containing ␺ bases were designed for study, a 9-bp complemen- at 5°C were referenced to the water resonance at 5.08 ppm. tary duplex (␺9 ϩ 9) consisting of the sequences (5Ј-GGU G␺A NOESY spectra of exchangeable protons were collected for each GUA-3Ј) and (5Ј-UAC UAC ACC-3Ј) and a model duplex sample by using excitation sculpting water-suppression tech- representing the branch-site U2 snRNA–intron pairing from the niques (ES-NOESY; ref. 24). ES-rotating-frame Overhauser yeast spliceosome (␺9 ϩ 10) consisting of the sequences (5Ј- effect spectroscopy (ES-ROESY) experiments were performed Ϸ GGU G␺A GUA-3Ј) and (5Ј-UAC UAA CAC C-3Ј) (Fig. 4). with 150-ms mixing times and 3-kHz spin-lock fields (B1). The only difference between the two duplexes, shown as the Experiments featuring a CLEANEX-PM spin-lock (18) com- extra A (underlined) in ␺9 ϩ 10, denotes the branch-site posite pulse scheme (Fig. 3) then were applied to the same adenosine, which is the nucleophile in the first step of spliceo- samples. some-mediated splicing of pre-mRNA. The oligomers were Acquisition parameters for the CLEANEX-PM pulse se- obtained from Dharmacon Research (Boulder, CO; refs. 22 and quence were optimized for application to RNA as compared with 23) and deprotected according to company protocols. Samples proteins (18). CLEANEX-PM experiments were performed were ethanol-precipitated, partially desalted by three washes of with mixing times of 5, 50, 100, 150, 200, 250, 300, 350, 500, 750, the pelleted RNA with 80% ethanol, and lyophilized to dryness.