Overextended RNA:DNA hybrid as a negative regulator of RNA polymerase II processivity.

An eight nucleotide RNA:DNA hybrid at the 3' end of the transcript is required for the stability of the elongation complex (EC) of RNA polymerase II. A non-template DNA strand is not needed for the stability of the EC, which contains this minimal hybrid. Here, we apply a recently developed method for promoter-independent assembly of functional EC of RNA polymerase II from synthetic RNA and DNA oligonucleotides to study the minimal composition of the nucleic acid array required for stability of the complex with RNA longer than eight nucleotides. We found that upon RNA extension beyond 14-16 nt in the course of transcription, non-template DNA becomes essential for maintaining a stable EC. Our data suggest that the overextended RNA:DNA hybrid formed in the absence the non-template DNA acts as a negative regulator of EC stability. The dissociation of the EC correlates with the backsliding of the polymerase along the overextended hybrid. The dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the EC and of RNA polymerase II processivity.

The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex.

The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.

Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3).

Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha(v)beta(3). Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I(2), which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha(IIb)beta(3)), but not by LM609 (anti-alpha(v)beta(3)), indicating that the interaction is mediated through integrin alpha(IIb)beta(3). In a solid phase binding assay, activated alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alpha(IIb)beta(3) failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin alpha(IIb)beta(3) on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth.

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate-early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin alphaVbeta3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an alphaV beta3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Adhesion of human umbilical vein endothelial cells to the immediate-early gene product Cyr61 is mediated through integrin alphavbeta3.

Cyr61 is a member of a family of growth factor-inducible immediate-early gene products thought to act cooperatively with the activities of growth factors. Upon synthesis, Cyr61 is secreted and is predominantly incorporated into the extracellular matrix. Recently, we demonstrated that Cyr61 promotes cell adhesion and migration and augments growth factor-induced DNA synthesis (Kireeva, M. L., Mo, F.-E., Yang, G. P., and Lau, L. F. (1996) Mol. Cell. Biol. 16, 1326-1334). In the present study, we investigated possible candidate receptor(s) on human umbilical vein endothelial cells (HUVECs) mediating adhesion to Cyr61. Under both serum-containing and serum-free conditions, adhesion of HUVECs to Cyr61 was dose-dependent, saturable, and abolished by affinity-purified anti-Cyr61 antibodies. Cell adhesion to Cyr61 was divalent cation-dependent and specifically inhibited by the peptide RGDS and LM609, a monoclonal antibody against integrin alphavbeta3. Furthermore, purified alphavbeta3 bound directly to an affinity matrix of Cyr61-coupled Sepharose 4B, and this interaction was specifically blocked by anti-Cyr61 antibodies. Additionally, in a solid phase binding assay, soluble Cyr61 bound to immobilized alphavbeta3 in a dose-dependent manner, and half-saturation binding occurred at approximately 5 nM Cyr61. As expected, the interaction of Cyr61 with immobilized alphavbeta3 was blocked by RGDS and LM609. In sum, these results identified Cyr61 as a novel ligand for alphavbeta3 and indicate that the adhesion of HUVECs to Cyr61 is mediated through interaction with this integrin. The possibility that integrin alphavbeta3 functions as a signaling receptor for Cyr61 accounts for most if not all activities that can be ascribed to Cyr61 to date and suggests a mechanism of action discussed herein.

Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development.

cyr61 and fisp12 are homologous immediate-early genes that are transcriptionally activated upon growth factor stimulation in fibroblasts. Their gene products belong to an emerging family of secreted proteins with a high degree of sequence homology, including conservation of all 38 cysteine residues in their secreted portions. We have recently shown that Cyr61 is an extracellular matrix (ECM) signaling molecule that promotes cell proliferation, migration, and adhesion. We describe herein the first purification of the Fisp12 protein and we compare the activities of purified Cyr61 and Fisp12, their metabolism, targeting, and their localization during development. Although Fisp12 is the mouse homolog of the human connective tissue growth factor (CTGF), it has no detectable mitogenic activity by itself. Rather, Fisp12 enhances fibroblast growth factor-induced DNA synthesis. The activities of Fisp12 and Cyr61 are nearly indistinguishable in three cell types tested: fibroblasts, endothelial, and epithelial cells. Both proteins are found in the ECM, although Cyr61 associates with the ECM more strongly and binds heparin with higher affinity. Fisp12, but not Cyr61, is also found in the culture medium, suggesting that Fisp12 might be able to act at a distance from its site of secretion, whereas Cyr61 might act more locally. Both secreted proteins are internalized and degraded through the lysosomal pathway, suggesting interaction with cell surface receptors. Both Cyr61 and Fisp12 are found in the placenta and the circulatory system as detected by immunohistochemistry, whereas Cyr61, but not Fisp12, is found in the skeletal and nervous systems. Fisp12, but not Cyr61, is found in secretory organs. Taken together, we propose that Cyr61 and Fisp12 are both signaling cell adhesion molecules that have similar or overlapping activities, and their differential sites of localization and targeting may dictate specificity in their biological roles.

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells.

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.

Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion.

cyr61 was first identified as a growth factor-inducible immediate-early gene in mouse fibroblasts. The encoded Cyr61 protein is a secreted, cystein-rich heparin-binding protein that associates with the cell surface and the extracellular matrix, and in these aspects it resembles the Wnt-1 protein and a number of known growth factors. During embryogenesis, cyr61 is expressed most notably in mesenchymal cells that are differentiating into chondrocytes and in the vessel walls of the developing circulatory system. cyr61 is a member of an emerging gene family that encodes growth regulators, including the connective tissue growth factor and an avian proto-oncoprotein, Nov cyr61 also shares sequence similarities with two Drosophila genes, twisted gastrulation and short gastrulation, which interact with decapentaplegic to regulate dorsal-ventral patterning. In this report we describe the purification of the Cyr61 protein in a biologically active form, and we show that purified Cyr61 has the following activities: (i) it promotes the attachment and spreading of endothelial cells in a manner similar to that of fibronectin; (ii) it enhances the effects of basic fibroblast growth factor and platelet-derived growth factor on the rate of DNA synthesis of fibroblasts and vascular endothelial cells, although it has no detectable mitogenic activity by itself; and (iii) it acts as a chemotactic factor for fibroblasts. Taken together, these activities indicate that Cyr61 is likely to function as an extracellular matrix signaling molecule rather than as a classical growth factor and may regulate processes of cell proliferation, migration, adhesion, and differentiation during development.

Circularly permuted dihydrofolate reductase of E. coli has functional activity and a destabilized tertiary structure.

Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.

Novel data on interactions of elongation factor Ts.

Interactions of EF-Ts with EF-Tu at all steps of the elongation cycle were studied by limited trypsinolysis, gel-filtration, analytical centrifugation and fluorescence polarization techniques. It is shown that EF-Ts does not dissociate from EF-Tu after GDP to GTP exchange, but remains bound to the Aa-tRNA.EF-Tu.GTP complex up to GTP hydrolysis stage on the ribosome. The possible role of these interactions is discussed.

[Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]. / Faktor élongatsii EF-Ts vzaimodeistvuet s kompleksom aminoatsil-tRNK.EF-Tu.GTP.

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.