guanosine-diphosphate and 7-methyl-diguanosine-triphosphate

Eukaryotic 5' mRNA cap structures participate to the post-transcriptional control of gene expression before being released by the two main mRNA decay pathways. In the 3'-5' pathway, the exosome generates free cap dinucleotides (m7GpppN) or capped oligoribonucleotides that are hydrolyzed by the Scavenger Decapping Enzyme (DcpS) forming m7GMP. In the 5'-3' pathway, the decapping enzyme Dcp2 generates m7GDP. We investigated the fate of m7GDP and m7GpppN produced by RNA decay in extracts and cells. This defined a pathway involving DcpS, NTPs and the nucleoside diphosphate kinase for m7GDP elimination. Interestingly, we identified and characterized in vitro and in vivo a new scavenger decapping enzyme involved in m7GpppN degradation. We show that activities mediating cap elimination identified in yeast are essentially conserved in human. Their alteration may contribute to pathologies, possibly through the interference of cap (di)nucleotide with cellular function.

Topics: Acid Anhydride Hydrolases; Adenosine Triphosphate; Dinucleoside Phosphates; Endoribonucleases; Guanosine Diphosphate; HEK293 Cells; Humans; N-Glycosyl Hydrolases; Neoplasm Proteins; Nucleoside-Diphosphate Kinase; RNA Cap Analogs; RNA Caps; RNA Stability; RNA, Messenger; Saccharomyces cerevisiae Proteins

While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identified a decapping activity in HeLa cytoplasmic extracts that releases (7me)GDP from capped transcripts. Decapping is activated in extracts by the addition of (7me)GpppG, which specifically sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion. AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence-specific ARE-binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

Topics: AT Rich Sequence; Binding, Competitive; Cell Extracts; Cytoplasm; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; HeLa Cells; Humans; Models, Genetic; Peptide Initiation Factors; Poly A; Poly(A)-Binding Proteins; Regulatory Sequences, Nucleic Acid; RNA Caps; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Saccharomyces cerevisiae

We screened a human cDNA library for proteins that bind mRNA cap methyltransferase (MT) and isolated nuclear transporter importin-alpha (Impalpha). This direct association was confirmed by glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and nuclear colocalization. In gel shift assays, MT selectively bound RNA containing 5'-terminal GpppG, and binding was inhibited by GpppG and not by m(7)GpppC. Impalpha markedly enhanced MT binding to GpppG-RNA and stimulated MT activity. MT/RNA/Impalpha complexes were dissociated by importin-beta, which also blocked the stimulation of cap methylation by Impalpha. The presence of RanGTP but not RanGDP prevented these effects of importin-beta. These findings indicate that importins play a novel role in mRNA biogenesis at the level of cap methylation.

Topics: Cell Nucleus; Dinucleoside Phosphates; Guanosine Diphosphate; Guanosine Triphosphate; HeLa Cells; Humans; Karyopherins; Methylation; Methyltransferases; Nuclear Proteins; ran GTP-Binding Protein; Recombinant Fusion Proteins; RNA Caps; Structure-Activity Relationship

The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand, and consists of a curved, 8-stranded antiparallel beta-sheet, backed by three long alpha-helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. Additional protein-ligand interactions include salt bridges and hydrogen bonds, plus water-mediated hydrogen bonds. The observed mode of 5' m-RNA cap recognition is almost certainly conserved among all known eIF4Es.

Topics: Crystallography, X-Ray; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Peptide Initiation Factors; Protein Conformation; Protein Structure, Secondary; RNA Caps