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...

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5 ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem-loop structures from one RNA form highly stringent crossstrand loop-loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions. retroviral RNA dimer ͉ RNA folding ͉ Selective 2Ј-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) chemistry ͉ site-directed cleavage R etroviral genomes usually consist of two sense-strand RNAs that are noncovalently linked near their 5Ј ends to form a dimeric structure (1-3). Recognition of this dimeric state ensures that exactly two RNA genomes are packaged into each nascent virion (1, 2). Mature retroviral virions contain almost exclusively retroviral genomic RNA plus a few select cellular RNAs (4, 5). Many other cellular RNAs, including mRNAs (6-8), are accessible to the retroviral packaging process. Specific recognition of retroviral genomic RNA against a large background of cellular RNA thus represents a striking example of molecular recognition in biology.We have recently identified a minimal dimerization active sequence (MiDAS) (9) for a representative gamma retrovirus, the Moloney murine sarcoma virus (MuSV; Fig. 1A). The MiDAS domain correlates closely with retroviral genomic sequences sufficient to package heterologous RNAs into virions (6,8,11,12), as dimers (8). The MiDAS domain also includes conserved sequence elements previously proposed to specify the noncovalent interactions that mediate RNA dimerization.Conserved sequence elements include self-complementary (palindromic) sequences (PAL1 and PAL2) and stem-loop structures 1 and 2 (SL1 and SL2) (10, 13-16). SL1 and SL2 contain GACG tetraloops that form stable loop-loop interactions with a second RNA molecule. Loop-loop interactions are mediated by canonical intermolecular C-G base pairing and additional stacking and intraand intermolecular hydrogen bonds (17) (see Fig. 6, which is published as supporting information on the PNAS web site). In addition, the self-complementary PAL1 and PAL2 sequences form extended heteroduplexes involving both strands in the dimer (refs. 13-16; C.S.B. and K.M.W., unpublished data). However, the kine...

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.

Ribose 2'-amine substitutions are broadly useful as structural probes in nucleic acids. In addition, structure-selective chemical reaction at 2'-amine groups is a robust technology for interrogating local nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for several RNA duplexes containing 2'-amino cytidine (C(N)) residues that form either C(N)-G base pairs or C(N)-A mismatches. The 2'-amine substitution is readily accommodated in an A-form RNA helix and thus differs from the C2'-endo conformation observed for free nucleosides. The 2'-amide product structure was visualized directly by acylating a C(N)-A mismatch in intact crystals and is also compatible with A-form geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in which the amine nucleophile carries a positive partial charge, we analyzed crystals of the C(N)-A duplex at pH 5, where the 2'-amine is protonated. The protonated amine moves to form a strong electrostatic interaction with the 3'-phosphodiester. Taken together with solution-phase experiments, 2'-amine acylation is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3'-phosphodiester group.

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