From primary sequence to static and dynamic RNA tertiary structures

RNA molecules fold back on themselves to form complex secondary and tertiary structures. Knowledge of the three-dimensional fold as well as the underlying dynamics is essential to understand sequence-function relationships in RNA. A novel technique, called SHAPE chemistry, has been created for determining RNA secondary structure. In this work, I expand the applications of SHAPE chemistry in two directions: calculating opening rates for slow moving nucleotides and analysis of RNA dynamics at nucleotide resolution. First, I use SHAPE to identify a novel class of slow-moving nucleotides and calculate their opening rates. The major mechanism is that certain C2[prime]-endo nucleotides have slow, seconds long, opening rates. Second, I find a strong correlation between SHAPE chemistry and molecular order as measured by 13C NMR relaxation experiments. This finding validates SHAPE as an adequate substitute for 13C NMR relaxation experiments, expanding nucleotide-resolution dynamics analysis to large RNAs, inaccessible to NMR methods. Finally, I develop a fast, fully automated modeling method to determine three-dimensional structures of RNAs. This method blends (1) SHAPE-derived secondary structure information; (2) biochemical experiments that yield high quality tertiary structure information with (3) a coarse-grained molecular dynamics refinement. It therefore does not require any prior assumptions about secondary or tertiary structure, establishing the foundation for modeling and refining large RNAs inaccessible to crystallography and NMR techniques

Strong Correlation between SHAPE Chemistry and the Generalized NMR Order Parameter ( S 2 ) in RNA

The functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2'-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo

Native-like RNA Tertiary Structures Using a Sequence-Encoded Cleavage Agent and Refinement by Discrete Molecular Dynamics

The difficulty of analyzing higher order RNA structure, especially for folding intermediates and for RNAs whose functions require domains that are conformationally flexible, emphasizes the need for new approaches for modeling RNA tertiary structure accurately. Here, we report a concise approach that makes use of facile RNA structure probing experiments that are then interpreted using a computational algorithm, carefully tailored to optimize both the resolution and refinement speed for the resulting structures, without requiring user intervention. The RNA secondary structure is first established using SHAPE chemistry. We then use a sequence-directed cleavage agent, that can be placed arbitrarily in many helical motifs, to obtain high quality inter-residue distances. We interpret this in-solution chemical information using a fast, coarse grained, discrete molecular dynamics engine in which each RNA nucleotide is represented by pseudoatoms for the phosphate, ribose and nucleobase groups. By this approach, we refine base paired positions in yeast tRNAAsp to 4 Å RMSD without any preexisting information or assumptions about secondary or tertiary structures. This blended experimental and computational approach has the potential to yield native-like models for the diverse universe of functionally important RNAs whose structures cannot be characterized by conventional structural methods

Slow Conformational Dynamics at C2′-endo Nucleotides in RNA

RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function

Wnt1 is a proangiogenic molecule, enhances human endothelial progenitor function, and increases blood flow to ischemic limbs in a HGF-dependent manner

Human endothelial progenitor cells (hEPCs) participate in neovascularization of ischemic tissues. Function and number of hEPCs decline in patients with cardiovascular disease, and therapeutic strategies to enhance hEPC function remain an important field of investigation. The Wnt signaling system, comprising 19 lipophilic proteins, regulates vascular patterning in the developing embryo. However, the effects of Wnts on hEPCs and the adult vasculature remain unclear. We demonstrate here that Wnt1 is expressed in a subset of endothelial cells lining the murine embryonic dorsal aorta and is reactivated in malignant angiosarcoma, suggesting a strong association of Wnt1 with angiogenesis. We investigate the effects of Wnt1 in enhancing hEPC function and blood flow to ischemic tissues and show that Wnt1 enhances the proliferative and angiogenic functions of hEPCs in a hepatocyte growth factor (HGF)-dependent manner. Injection of Wnt1-expressing hEPCs increases blood flow and capillary density in murine ischemic hindlimbs. Furthermore, injection of Wnt1 protein alone similarly increases blood flow and capillary density in ischemic hindlimbs, and this effect is associated with increased HGF expression in ischemic muscle. These findings demonstrate that Wnt1, a marker of neural crest cells and hitherto unknown angiogenic function, is a novel angiogenic protein that is expressed in developing endothelial cells, exerts salutary effects on postnatal hEPCs, and can be therapeutically deployed to increase blood flow and angiogenesis in ischemic tissues.—Gherghe, C. M., Duan, J., Gong, J., Rojas, M., Klauber-Demore, N., Majesky, M., Deb, A. Wnt1 is a proangiogenic molecule, enhances human endothelial progenitor function, and increases blood flow to ischemic limbs in a HGF-dependent manner