Nancy L. Greenbaum

Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2′ OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5′ splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (ψ) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this ψ in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The ψ-induced structure places the nucleophile in an accessible position for the first step of splicing.

Investigation of Overhauser effects between pseudouridine and water protons in RNA helices

The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (ψ), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uridine. When ψ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The presence of ψ 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 ψNH1 groups and nearby phosphate 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. (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for ψNH1–water cross-relaxation effects within two RNA helices: (i) a complementary duplex, in which ψ 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 conserved ψ extrudes the branch-site adenosine from the helix. Our data implicate a water–ψNH1 hydrogen bond both in stabilizing the complementary helix and in favoring formation of the unique structure of the branch-site helix.

Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2–U6 snRNA complex

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2–U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

The electrostatic characteristics of G · U wobble base pairs

G·U wobble base pairs are the most common and highly conserved non-Watson–Crick base pairs in RNA. Previous surface maps imply uniformly negative electrostatic potential at the major groove of G·U wobble base pairs embedded in RNA helices, suitable for entrapment of cationic ligands. In this work, we have used a Poisson–Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G·U wobble displays distinctly enhanced negativity compared with standard GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G·U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5 kT/e, depending upon structural features. We propose that the negativity at the major groove of G·U wobble base pairs is determined by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G·U sites in RNA are most likely to bind cationic ligands.

DNA damage-site recognition by lysine conjugates

Simple lysine conjugates are capable of selective DNA damage at sites approximating a variety of naturally occurring DNA-damage patterns. This process transforms single-strand DNA cleavage into double-strand cleavage with a potential impact on gene and cancer therapy or on the design of DNA constructs that require disassembly at a specific location. This study constitutes an example of DNA damage site recognition by molecules that are two orders of magnitude smaller than DNA-processing enzymes and presents a strategy for site-selective cleavage of single-strand nucleotides, which is based on their annealing with two shorter counterstrands designed to recreate the above duplex damage site.

Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2′-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosomes catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958–965] showed that a phylogenetically conserved pseudouridine (ψ) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosomes branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of ψ-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2′-OH, in the ψ-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson–Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935–955] revealed a region of exceptionally negative potential in the major groove surrounding the 2′-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10-50 Å), mid-range (50-150 Å), and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.

Specificity of Mg2+ binding at the Group II intron branch site

Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg(2+) and Ca(2+) still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg(2+) may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.

Nanomaterials: hammerheading a new frontier

Energy transfer from organic fluorophores to small metal nanoparticles is being used as a molecular beacon tool to monitor the kinetic processes of the hammerhead ribozyme. This marks the first time that nanomaterials have been used to monitor ribozyme kinetics. The quantum efficiency of energy transfer from the fluorophore to the gold nanoparticle follows a distance dependence behavior, which allows the real-time characterization of ribozyme complex structure and cleavage kinetics. The rate of cleavage for our ribozyme at pH=6.5 and 37°C is measured to be on the order of 10-2 min-1, which is the correct order of magnitude for similar ribozymes at this pH in the literature.

Interaction between the Spliceosomal Pre-mRNA Branch Site and U2 snRNP Protein p14.

We have probed the molecular basis of recognition between human spliceosomal U2 snRNP protein p14 and RNA targets representing the intron branch site region. Interaction of an RNA duplex representing the branch site helix perturbed at least 10 nuclear magnetic resonance cross-peaks of (15)N-labeled p14. However, similar chemical shift changes were observed upon interaction with a duplex without the bulged branch site residue, suggesting that binding of p14 to RNA is nonspecific and does not recognize the branch site. We propose that the p14-RNA interaction screens charges on the backbone of the branch site during spliceosome assembly.