Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.

mRNA degradation predominantly proceeds through two alternative routes: the 533 pathway, which requires deadenylation followed by decapping and 533 hydrolysis; and the 335 pathway, which involves deadenylation followed by 335 hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp 3 G, m 7 Gp 3 G, or m 2 7,3-O Gp 3 G) and 3 termini (A 31 , A 60 , or G 16 ) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5 terminus to bind eIF4E, not merely the presence of a blocking group at the 5-end. Introducing a stem-loop in the 5-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. The 5Ј terminus of all eukaryotic cellular mRNAs is modified with a 5Ј-5Ј m 7 GTP-containing cap (1). Caps fulfill a variety of functions in the synthesis, translation, and degradation of mRNA. The presence of the 5Ј cap structure increases both the accuracy and efficiency of pre-mRNA splicing (2, 3). The cap on pre-mRNA interacts with the nuclear cap-binding complex, which remains bound and plays an active role during RNA processing and export (4). In the cytosol, the cap structure is required for efficient translation of mRNA. The cap is specifically recognized by the translational initiation factor eIF4E (5, 6). Binding of eIF4E to the cap occurs during formation of the 48 S initiation complex, which is rate limiting for translation initiation under normal conditions (7,8). Finally, the cap serves as one determinant of mRNA degradation. Capped mRNAs are more stable than their uncapped counterparts (9). The cap structure helps to protect RNA from degradation by 5Ј33Ј-exonucleases located in the cytosol and nucleus, as demonstrated in both Saccharomyces cerevisiae (10) and mammalian cells (11,12).A second stability element in mRNA is the 3Ј-terminal poly(A) tract. PABP 2,3 binds to poly(A) and is essential for the stability provided by this element, protecting mRNA against exonucleolytic degradation (12-15). PABP also binds to the N terminus of eIF4G (16) and stabilizes the eIF4G⅐eIF4E complex, enhancing translational reinitiation (17, 18). The stimulation conferred by the cap and poly(A) tract are synergistic rather than additive (19,20). Thus, for both translation and degradation of mRNA, elements binding to the 5Ј and 3Ј termini act cooperatively and in close proximity.There are two major pathways by which polyadenylated mRNA is degraded in eukaryotic cells, a 5Ј33Ј pathway and a 3Ј35Ј pathway, as well as two specialized routes for aberrant mRNA degradation (21). In both the 5Ј33Ј and 3Ј35Јpathways, shortening of the poly(A) tract initiates mRNA decay. There are several mechanism of deadenylation (21), but one of them involves a poly(A)-specific ribonuclease (22), an enzyme tha...

A general and efficient method for the microwave-assisted formation of the C-P bond was developed. Using a prevalent palladium catalyst, Pd(PPh3)4, a quantitative cross-coupling of various H-phosphonate diesters with aryl and vinyl halides was achieved in less than 10 min. The reactions occurred with retention of configuration at the phosphorus center and in the vinyl moiety. Using this protocol, several C-phosphonates, including those bearing nucleoside and cholesteryl moieties, were prepared in high yields.

An efficient protocol for the synthesis of arylphosphonate diesters via a palladium-catalyzed cross-coupling of H-phosphonate diesters with aryl electrophiles, promoted by acetate ions, was developed. A significant shortening of the cross-coupling time in the presence of the added acetate ions was achieved for bidentate and monodentate supporting ligands, and for different aryl electrophiles (iodo, bromo and triflate derivatives). The reaction conditions were optimized in terms of amount of the catalyst, supporting ligands, and source of the acetate ion used. Various arylphosphonates, including those of potential biological significance, were synthesized using this newly developed protocol. Some mechanistic aspects of the investigated reactions are also discussed.

Pd(PPh 3 ) 4 , Pd(dba) 2 , Pd(OAc) 2 , and PdCl 2 , have been evaluated as possible Pd(0) sources for the palladium-catalyzed P-C bond formation via a cross-coupling of aryl halides with H-phosphonate diesters. It was found that the most efficient catalytic system can be generated from Pd(OAc) 2 with a key role being played by Pd(II) and Pd(0) species with coordinated acetate ions. The reactivity of differently ligated Pd(II) complexes was determined, and 31 P NMR spectroscopy studies were carried out to provide mechanistic interpretations for the observed differences between the catalytic systems.

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DFT calculations are used to elucidate the reaction mechanism, the role of the chlorosilyl group, and primary vs. secondary and C(sp3)–H vs. C(sp2)–H selectivity of the iridium-catalyzed borylation of chlorosilanes.

Ligand substitution and reductive elimination of the palladium-catalyzed C−P bond forming cross-coupling were investigated in depth. It was found that for PhPdII(PPh3)2X (X = I, Br, Cl) complexes, a step commonly referred to as ligand substitution commenced with coordination of an H-phosphonate diester, followed by its deprotonation to form an equilibrium mixture of penta- and tetracoordinate palladiumphosphonate intermediates, from which reductive elimination of the product (diethyl phenylphosphonate) occurred. For the acetate counterpart, PhPdII(PPh3)2(OAc), the incorporation of a phosphonate moiety to the complex was preceded by a rate-determining removal of the supporting phosphine ligand, facilitated by an intramolecular catalysis by the acetate group. Both the reaction steps, i.e., formation of palladiumphosphonate intermediates and reductive elimination, were significantly faster for the acetate versus halides containing PdII complexes investigated. Similar observations were found to be true also for bidentate ligand complexes [(dppp)PdII(Ph)X]; however, in this instance, a single palladiumphosphonate intermediate, (dppp)PdII(Ph)(PO(OEt)2), could be observed by 31P NMR spectroscopy. The synthetic and kinetic studies on the cross-coupling reaction of diethyl H-phosphonate with phenyl halides permitted us to elucidate a crucial catalytic role of an acetate group in PdII complexes and to propose two distinctive catalytic cycles, which complemented traditional Pd0/PdII schemes, for the palladium-mediated C−P bond formation.