Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ‐wide in a Wnt‐dependent manner, expands, undergoes epithelial–mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro‐fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/βcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro‐fibrotic Wnt1/βcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury.

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.

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 tRNA Asp 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.

Local and global dynamics in folded RNAs occur over broad timescales spanning picoseconds to minutes. 1 Slow motions likely play predominant roles in governing RNA folding and ribonucleoprotein assembly reactions. However, slow local motions are extremely difficult to detect, especially for large RNAs with complex structures.The local environment and degree of flexibility can be evaluated at nucleotide resolution for RNAs of any size using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry. 2 RNA nucleotides exist in equilibrium between constrained (closed) and flexible (open) states. The 2′-OH group in flexible nucleotides preferentially adopts an open, reactive, conformation that facilitates reaction with electrophilic reagents to form a 2′-O-adduct (Figure 1). SHAPE experiments work well using electrophiles based on the isatoic anhydride (IA) scaffold. 2a,3 Positions that form 2′-O-adducts are detected by primer extension. 2 IA derivatives both react with the RNA 2′-OH group and also undergo concurrent degradation by hydrolysis (Figure 1). 2′-OH reactivity is thus conveniently monitored by allowing a reaction to proceed until the reagent has been consumed, either by hydrolysis or reaction with RNA. At this end point, the fraction adduct at any nucleotide (f) is (1) Where (2) and the rate of hydrolysis has been shown to be proportional to the rate of adduct formation, 2b,3 k adduct /k hydrolysis = β. These relationships lead to two limits. In limit 1, k open + k close ≫ k adduct [reagent] Correspondence to: Kevin M. Weeks, weeks@unc.edu. Supporting Information Available: Methods and four figures. This material is available free of charge via the Internet at http:// pubs.acs.org. HHS Public AccessIt should therefore be possible to monitor local nucleotide dynamics in RNA under conditions where limit 2 applies by varying the reactivity (or k hydrolysis ) of the hydroxylselective electrophile. IA has a hydrolysis half-life (t 1/2 ) of 430 s at 37 °C. Electronwithdrawing substituents at the cyclic amine (R 1 ) or in the benzene ring (R 2 ) enhance reagent reactivity. Compared to IA, N-methyl isatoic anhydride (NMIA), 4-nitroisatioc anhydride (4NIA), and 1-methyl 7-nitroisatoic anhydride (1M7) 3 have progressively shorter hydrolysis half-lives (table, Figure 1).To investigate if distinct local nucleotide dynamics can be captured by varying the SHAPE electrophile, we focused on an important variation in RNA structure: the C2′-endo conformation. Although C2′-endo nucleotides are relatively rare, they are highly overrepresented in important RNA tertiary interactions and in catalytic active sites. 4 Local structure at tandem G•A mismatches depends on the local sequence context. 5 Guanosine nucleotides in G•A pairs adopt the C2′-endo conformation in the sequences (UGAA) 2 5a and (GGAU) 2 , 5b the C3′-endo conformation typical of standard A-form helix geometry in (CGAG) 2 , 5c and a mixture of C2′-endo/C3′-endo conformations in (UGAG) 2 . 5b We constructed a simple hairpin RNA (termed...