Formation of a complex of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin with G-quadruplex DNA

A water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TmPyP4), has been studied extensively because of its unique physicochemical properties that lead to interactions with nucleic acids, as well as its therapeutic application. Formation of a complex between TmPyP4 and parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized in an effort to elucidate the mode of molecular recognition between TmPyP4 and the DNA. The study demonstrated that TmPyP4 intercalates into the A3pG4 step of [d(TTAGGG)]4 with an association constant of 6.2 x 10(6) M(-1) and a stoichiometric ratio of 1:1. The binding of TmPyP4 to the A3pG4 step of [d(TTAGGG)]4 was found to be stabilized by the pi-pi stacking interaction of the porphyrin ring of TmPyP4 with the G4 quartet as well as the A3 bases of the G-quadruplex DNA. These findings provide novel insights for the design of porphyrin derivatives that bind to DNA with high affinity and specificity.     

Expression and localization of transcripts of MT5-MMP and its related MMP in the ovary of the medaka fish Oryzias latipes

cDNA clones of MT5-matrix metalloproteinase (MT5-MMP) and a related protein (designated MT5-MMP-del) were isolated by screening the cDNA library and by 3'-rapid amplification of cDNA ends using an ovary RNA of the medaka fish Oryzias latipes. The MT5-MMP clone encodes a protein of 546 amino acids while the MT5-MMP-del clone encodes a protein of 431 amino acids. Compared with mammalian counterparts, the fish MT5-MMP and MT5-MMP-del both lack the signal peptide and a part of the prodomain. The fish MT5-MMP and MT5-MMP-del were different in that the latter did not have the stem/transmembrane/cytoplasmic domain. The two fish MMPs were expressed in the ovary, testis, brain, and intestine. In the ovary, MT5-MMP mRNA was expressed in the oocytes of small growing follicles. In contrast, MT5-MMP-del mRNA was found in the stromal interstitial cells. These results strongly suggest that a MT5-MMP/gelatinase A cascade may possibly operate in the process of spawning and/or other events associated with ovulated oocytes or fertilized eggs of the medaka fish.     

Translational repression of the disintegrin and metalloprotease ADAM10 by a stable G-quadruplex secondary structure in its 5'-untranslated region

Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-β peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.     

Contrasting expression of membrane metalloproteinases, MT1-MMP and MT3-MMP, suggests distinct functions in skeletal development

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.     

Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.     

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.     

The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.     

Connexin43 mRNA contains a functional internal ribosome entry site

A reporter gene construct was used to study the regulation of connexin43 (Cx43) expression, the major gap junction protein found in heart and uterus, in transfected cell lines. The construct had the firefly luciferase gene under the control of the Cx43 promoter. Inclusion of the 5'-untranslated region (UTR) of the mRNA in the construct increased luciferase expression by 70%. A bicistronic vector assay demonstrated that the Cx43 5'-UTR contains a strong internal ribosome entry site (IRES). Deletion analysis localized the IRES element to the upstream portion of the 5'-UTR.