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Enzymology of RNA cap synthesis

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Abstract The 5′ guanine‐N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co‐transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7‐methyl guanosine linked through a 5′‐5′ inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co‐transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway. Copyright © 2010 John Wiley & Sons, Ltd. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Processing > tRNA Processing

Structures of yeast, viral, and metazoan RNA triphosphatases. (a) Structure of homodimeric metal‐dependent RNA triphosphatase Cet1 (PDB 1D8H) from S. cerevisiae with a view looking into the triphosphate tunnels. The structure is depicted in ribbon representation with loops and α‐helices colored gray and β‐strands colored salmon. The divalent cation manganese and sulfate ion are labeled and depicted in ball‐and‐stick representation. Basic and acidic residues that point into the tunnel are shown in sticks. Amino‐ and carboxy‐termini are denoted ‘N’ and ‘C’, respectively. Disordered segments are shown as dashed lines. (b) Structure of the monomeric RNA triphosphatase domain (amino acid 1–237) of mimivirus capping enzyme, MimiCE (PDB 3BGY) colored and structurally depicted as in (a). (c) The RNA triphosphatase domain of mammalian mRNA capping enzyme Mce1 (PDB 1I9S). Ribbon representation colored pink with the active site cysteine (Cys126) denoted in ball‐and‐stick representation and labeled. Structural graphics prepared using PyMOL (http://pymol.sourceforge.net/).

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Mechanisms of cap 0 synthesis. Triphosphatase activities: Metal‐dependent (top) or metal‐independent RNA triphosphatases (bottom) catalyze removal of the γ‐phosphate (colored red) from pppRNA to generate ppRNA and release of inorganic phosphate (colored black). The tunnel architecture of the metal‐dependent RNA triphosphatase Cet1 indicating side chains that coordinate pppRNA and two metals, one (solid sphere) derived from structural studies and the other (circle containing M) derived from biochemical studies. Metal‐independent RNA triphosphatases catalyze removal of the γ‐phosphate through a two‐step reaction through a covalent protein‐cysteinyl‐S‐phosphate intermediate (step 1, shown in the figure) colored as in top panel. Guanylyltransferase activities: RNA guanylyltransferase catalyzes capping in a two‐step reaction. In step 1, the enzyme binds GTP (colored blue) and magnesium (not shown) to catalyze transfer of GMP to the active site lysine to form a covalent enzyme(lysyl‐N)‐GMP intermediate. In step 2, the enzyme binds the ppRNA (colored red) to catalyze transfer of the GMP to form GpppRNA. Methyltransferase activities: RNA guanine‐N7 methyltransferase binds S‐adenosylmethionine (AdoMet) (colored green) and GpppRNA (colored as above) to catalyze transfer of the methyl group (colored green) to the guanine N7 position.

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Structures of viral and yeast RNA guanylyltransferases. (a) Structure of Chlorella virus capping enzyme PbCV‐1 in ‘open’ configuration (PDB 1CKN) depicting the nucleotidyltransferase (NTase) domain (blue) and OB domain (yellow) bound to GTP (labeled). The active site lysine (Lys82) and GTP are shown in stick representation. The position of the OB‐fold domain (green) in the ‘closed’ conformation was superimposed on the ‘open’ conformation based on alignment of the respective NTase domains. (b) Structure of the Candida albicans RNA guanylyltransferase, Cgt1 (PDB 1P16; chain A). The covalent lysyl‐GMP adduct is represented in stick with NTase (blue) and OB‐fold (yellow) domains shown in ribbon representation. View of Cgt1 generated by aligning its NTase domain to that in (a). OB‐fold domains of (c) C. albicans Cgt1 and (d) S. cerevisiae Ceg1 (PDB 3KYH) to illustrate conserved OB elements (yellow) and insertion elements (salmon) that were shown to contribute to interactions with the triphosphatase. Disordered regions are represented by dashed lines.

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Structures and synthesis of RNA caps. Cap 0 or m7G caps (GMP colored blue) are formed in sequential steps by three enzymatic activities that act on the 5′ triphosphate end (colored red) of nascent transcripts. Transfer of a methyl group (colored green) from S‐adenosylmethionine (AdoMet) completes the synthesis of cap 0. Transfer of two methyl groups (colored magenta) from S‐adenosylmethionine (AdoMet) is required to form the TMG cap. Caps 1 and 2 structures require methylation (colored magenta) of cap 0 at the ribose 2′‐O at the first and second nucleosides, respectively. Cap 4 structure is generated by six rounds of methylation (colored magenta). The first three rounds of methylation (colored magenta) occur at two positions on the base and ribose of the first nucleoside of the primary transcript. The next three rounds of methylation (colored magenta) occur on ribose 2′‐O positions of the next three nucleosides. The γ‐methyl phosphate cap is formed by transfer of a methyl group (colored magenta) from AdoMet to the γ‐phosphate of the primary transcript.

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Unconventional pathways for cap formation. (a) Schematic of the proposed mechanism of mRNA cap formation in rhabdovirus with protein colored purple, catalytic histidine residue denoted in red, nascent mRNA colored red, terminal phosphates of nascent mRNA and phosphatases of the guanine nucleotide shown as yellow balls with phosphate positions labeled. (b) Schematic of influenza virus ‘cap snatching’ indicating the host capped mRNA (blue), the viral RNA (red), the PB2 subunit (green), the PA subunit (orange), and the PB1 subunit (colored in cyan). (c) Schematic of the proposed pathway for capping in alphavirus. The nsP1 protein (pink) transfers a methyl group (green) from AdoMet (black) to GTP (blue and orange spheres) to generate N7‐methyl GTP. The N7‐methyl GTP is then used as a substrate to cap the viral RNA (red).

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Schematic models for co‐transcriptional mRNA capping in S. cerevisiae, S. pombe and mammals. In S. cerevisiae, the RNAP II CTD (light‐green) is shown hyperphosphorylated at Ser2 and Ser5 positions (gold spheres with P). The RNA guanylyltransferase interacting with the phosphorylated CTD via the NTase domain (blue) and Cet1 (magenta) via the OB domain (yellow). Cet1 and Ceg1 are tethered by a flexible linker (dashed magenta line). The RNA guanine‐N7 methyltransferase Abd1 (green) shown independently interacting with the phosphorylated RNAP II CTD. In S. pombe, the RNA triphosphatase Pct1 (magenta), RNA guanylyltransferase Pce1 (NTase blue, OB yellow), and RNA guanine‐N7 methyltransferase Pcm1 (green) are shown interacting independently with the phosphorylated RNAP II CTD. The bifunctional mammalian capping enzyme shown with the N‐terminal RNA triphosphatase domain (pink) and RNA guanylyltransferase domain linked by a flexible tether (dashed line) with the RNA guanylyltransferase NTase domain (blue) and OB domain (yellow). Interactions between the bifunctional mammalian capping enzyme and phosphorylated RNAP II CTD are mediated by the NTase domain. The mammalian RNA guanine‐N7 methyltransferase (green) shown interacting independently with the phosphorylated RNAP II CTD. The nascent mRNA is denoted by red lines in each of the panels with the triphosphate terminus indicated by gold spheres.

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Structures of the capping apparatus from S. cerevisiae and bluetongue virus and a complex between the phosphorylated RNAP II CTD and C. albicans guanylyltransferase. (a) Structure of a complex between S. cerevisiae Cet1 and Ceg1. The Cet1 homodimer is shown with Cet1 protomers colored cyan and magenta. The Ceg1 NTase domain is colored blue and OB domain is colored yellow. Dashed lines indicate disordered amino acids. The Cet1 WAQKW motifs are represented in ball‐and‐stick and specified amino acid side chains are labeled. Lys70 is shown in Ceg1 to indicate the position of the guanylyltransferase active site. (b) Structure of bluetongue virus VP4 (PDB 2HJA) in ribbon representation indicating the following domains: kinase‐like (KL; gold), N7‐methyltransferase (N7 MTase; green), 2′‐O methyltransferase (2′‐O MTase; gray), and triphosphatase/nucleotidyltransferase (TPase/NTase; blue). GpppG shown in the 2′‐O MTase active site with S‐adenosyl‐homocysteine (AdoHcy, PDB 2HJP) shown in both N7 and 2′‐O MTase active sites in stick representation. Dashed lines indicated disordered regions. (c) The structure of C. albicans Cgt1 (PDB 1P16; chain B) with NTase (blue), OB domain (yellow) bound to a Ser5 phosphorylated RNAP II CTD peptide (green). Close‐up view of the CTD binding site with positions for Tyrosine 1, Proline 3, and phosphorylated Ser5 indicated. The CTD and GTP in the active site shown in stick representation. Dashed lines denote disordered regions.

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Schematic diagram depicting various architectures of the capping apparatus. Domain organizations in different taxa showing the tunnel shaped metal‐dependent RNA triphosphatase (TPase; pink with white interior), the nucleotidyltransferase (NTase; blue), the OB‐fold (OB; yellow), and guanine‐N7 methyltransferase (N7‐MTase; green). For ISKNV only the metal‐independent TPase and NTase domains are shown, as the virus is not known to encode an N7‐MTase. For the bluetongue virus capping enzyme, the KL domain (gold) and 2′‐O MTase (gray) are also included. Domains are shown as boxes, catalytic residues are labeled and indicated by red vertical lines, and flexible linkers between domains denoted by dashed lines.

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Structures of viral, protozoan, and metazoan guanine‐N7 methyltransferases. (a) Orthogonal views of vaccinia virus guanine‐N7 methyltransferase (PDB 2VDW) depicting the D1 catalytic subunit (marine) and D12 regulatory subunit (yellow) in ribbon diagram. S‐adenosyl‐homocysteine (AdoHcy) and a sulfate ion in the D1 active site are represented in ball‐and‐stick. The N‐terminal peptide (red) that covers AdoHcy depicted as a ribbon and transparent molecular surface. Dashed lines denote disordered regions of the structure. (b) Structure of RNA guanine‐N7 methyltransferase from Encephalitozoon cuniculi, Ecm1 (PDB 1RI1) shown in a similar view to that presented for the D1 subunit in the left panel of (a). GTP and AdoHcy are shown in the Ecm1 active site in ball‐and‐stick. (c) Structure of human RNA guanine‐N7 methyltransferase Hcm1 (PDB 3EPP) aligned as in (b) with AdoHcy shown in ball‐and‐stick. Amino‐ and carboxy‐termini denoted as ‘N’ and ‘C’, respectively.

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