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mRNA maturation in giant viruses: variation on a theme.

Priet S, Lartigue A, Debart F, Claverie JM, Abergel C - Nucleic Acids Res. (2015)

Bottom Line: Unexpectedly, the vPAPs are homodimeric and uniquely self-processive.The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication.A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 7257, Aix-Marseille Université, 163 Avenue de Luminy, Case 932, 13288 Marseille cedex 9, France.

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Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN13), N7- or 2′O-methylated capped RNA substrates (7MeGpppAN13 or GpppA2′OMeN13), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.
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Figure 7: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN13), N7- or 2′O-methylated capped RNA substrates (7MeGpppAN13 or GpppA2′OMeN13), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

Mentions: Even though we postulated that a cap specific 2′O MTase is present in the conserved gene cluster encoding the mimiviruses mRNA capping enzyme, we wondered whether the Mg18, like its Vaccinia virus VP39 homologue, could retain the 2′O MTase activity (Supplementary Figure S1C, step 3). Sequence analysis revealed that Mg18 harbors the conserved catalytic K-D-K-E tetrad shared by all known 2′O MTases including that of poxvirus VP39 (33), flavivirus (54), coronavirus (55) and rhabdovirus (56). In the VP39 structure cap recognition is made by the stacking of two aromatic side chains (Y22 and a F180) and hydrogen bonding with two acidic side chains (D182, E233). The first aromatic residue is conserved in the Mg18 sequence (Y59) while the loop bearing the F180 and the D182 residues is 4 amino acids shorter in the Mg18 sequence suggesting that Mg18 may not be as specific for cap residues as VP39 (57). We compared the MTase activities of Mg18, VP39 and the Human N7 MTase (hN7 MTase) on several RNA substrates (Figure 7). First, Mg18 has a much greater activity on the capped GpppAN13 than the uncapped pppAN13, therefore is dependent upon 5′-guanylylation. In contrast to VP39, Mg18 had comparable activity with 7MeGpppAN13 and GpppAN13 and therefore, its activity does not dependent on the cap methylation status. Mg18 is about 10 times less efficient on 2′O-methylated capped RNAs (GpppA2′OMeN13) than on N7-methylated or unmethylated caps (7MeGpppAN13 or GpppAN13), indicating that the methylated residue in GpppA2′OMeN13 is the methylation target. As expected, the addition of Mg561 had no influence on the 5′-GpppA specific Mg18 MTase activity (Supplementary Figure S8). Then we investigated the methylation state of the A. castellanii polyA+ mRNA caps using VP39 and the hN7 MTase. The A. castellanii mRNAs were not methylated by the two MTases, suggesting that their caps were already methylated both at the N7 and at the ribose 2′O positions. To our surprise, Mg18 exhibited a strong MTase activity on the polyadenylated A. castellanii mRNAs (Figure 7), suggesting that Mg18 had a second distinct activity: the expected 5′-GpppA specific 2′O MTase activity, and another cap independent one. To investigate this additional function of Mg18, we assayed Mg18 activity on uncapped homopolymeric poly (A), (U), (C) and (G) substrates. We found an even stronger MTase activity of Mg18 on polyadenylates. In the absence of any cap structure, Mg18 is able to methylate the ribose 2′O position of internal nucleotides with a clear specificity for adenylates suggesting it can also methylate the mRNAs polyA tails (Supplementary Figure S1C, step 7).


mRNA maturation in giant viruses: variation on a theme.

Priet S, Lartigue A, Debart F, Claverie JM, Abergel C - Nucleic Acids Res. (2015)

Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN13), N7- or 2′O-methylated capped RNA substrates (7MeGpppAN13 or GpppA2′OMeN13), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.
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Figure 7: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN13), N7- or 2′O-methylated capped RNA substrates (7MeGpppAN13 or GpppA2′OMeN13), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.
Mentions: Even though we postulated that a cap specific 2′O MTase is present in the conserved gene cluster encoding the mimiviruses mRNA capping enzyme, we wondered whether the Mg18, like its Vaccinia virus VP39 homologue, could retain the 2′O MTase activity (Supplementary Figure S1C, step 3). Sequence analysis revealed that Mg18 harbors the conserved catalytic K-D-K-E tetrad shared by all known 2′O MTases including that of poxvirus VP39 (33), flavivirus (54), coronavirus (55) and rhabdovirus (56). In the VP39 structure cap recognition is made by the stacking of two aromatic side chains (Y22 and a F180) and hydrogen bonding with two acidic side chains (D182, E233). The first aromatic residue is conserved in the Mg18 sequence (Y59) while the loop bearing the F180 and the D182 residues is 4 amino acids shorter in the Mg18 sequence suggesting that Mg18 may not be as specific for cap residues as VP39 (57). We compared the MTase activities of Mg18, VP39 and the Human N7 MTase (hN7 MTase) on several RNA substrates (Figure 7). First, Mg18 has a much greater activity on the capped GpppAN13 than the uncapped pppAN13, therefore is dependent upon 5′-guanylylation. In contrast to VP39, Mg18 had comparable activity with 7MeGpppAN13 and GpppAN13 and therefore, its activity does not dependent on the cap methylation status. Mg18 is about 10 times less efficient on 2′O-methylated capped RNAs (GpppA2′OMeN13) than on N7-methylated or unmethylated caps (7MeGpppAN13 or GpppAN13), indicating that the methylated residue in GpppA2′OMeN13 is the methylation target. As expected, the addition of Mg561 had no influence on the 5′-GpppA specific Mg18 MTase activity (Supplementary Figure S8). Then we investigated the methylation state of the A. castellanii polyA+ mRNA caps using VP39 and the hN7 MTase. The A. castellanii mRNAs were not methylated by the two MTases, suggesting that their caps were already methylated both at the N7 and at the ribose 2′O positions. To our surprise, Mg18 exhibited a strong MTase activity on the polyadenylated A. castellanii mRNAs (Figure 7), suggesting that Mg18 had a second distinct activity: the expected 5′-GpppA specific 2′O MTase activity, and another cap independent one. To investigate this additional function of Mg18, we assayed Mg18 activity on uncapped homopolymeric poly (A), (U), (C) and (G) substrates. We found an even stronger MTase activity of Mg18 on polyadenylates. In the absence of any cap structure, Mg18 is able to methylate the ribose 2′O position of internal nucleotides with a clear specificity for adenylates suggesting it can also methylate the mRNAs polyA tails (Supplementary Figure S1C, step 7).

Bottom Line: Unexpectedly, the vPAPs are homodimeric and uniquely self-processive.The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication.A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 7257, Aix-Marseille Université, 163 Avenue de Luminy, Case 932, 13288 Marseille cedex 9, France.

Show MeSH
Related in: MedlinePlus