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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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Mg561 and R341 are processive PolyA polymerases. (A) Time course (in minutes) PAP assay of Mg561, R341, Mg18 and a mixture of Mg18 with Mg561 or R341, on a 20-mers 5′-32P-labeled RNA primer with Mg2+ as catalytic ions. (B) As in (A), with Mn2+ instead of Mg2+ (C) Catalytic activity of Mg561 on each four nucleotides on a 20-mers and Mn2+.
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Figure 2: Mg561 and R341 are processive PolyA polymerases. (A) Time course (in minutes) PAP assay of Mg561, R341, Mg18 and a mixture of Mg18 with Mg561 or R341, on a 20-mers 5′-32P-labeled RNA primer with Mg2+ as catalytic ions. (B) As in (A), with Mn2+ instead of Mg2+ (C) Catalytic activity of Mg561 on each four nucleotides on a 20-mers and Mn2+.

Mentions: To confirm the functional prediction of Mg561 and R341, the proteins were purified and assayed in an in vitro polyadenylation assay on a synthetic 20-mers RNA without secondary structure in the presence of ATP and Mg2+ or Mn2+ as catalytic ions (Figure 2A and B). The two proteins were able to synthesize long products, apparently proccessively, derived from the RNA and ATP, in the presence of each cation, demonstrating the intrinsic property of Mg561 and R341 to polyadenylate RNA in absence of any specific signal (Supplementary Figure S1C, step 6). This in turn suggested that in vivo the hairpin recognition and processing could require additional proteins (Supplementary Figure S1C, step 5). In contrast to the Vaccinia and ASFV PAPs which stop after the addition of ∼30 adenylates (16,48), Mg561 and R341 were able to add much longer polyA tails (up to 700 adenylates). The nucleotide specificity of the PAPs was then assayed in the presence of Mn2+ and the 4 NTPs (Figure 2C). As expected, ATP was by far the most efficient nucleotide for homopolymeric tail synthesis. Similar results were obtained when using Mg2+ instead of Mn2+ (data not shown). However, as observed for non-canonical cellular PAPs known as terminal uridyltransferases (TUTase) or polyU polymerases (PUP) (49), Mg561 was also able to add long polyU tails, although with a reduced activity. This property was also reported for Vaccinia VP55 (29). This enzyme requires uridylates 30–40 nt upstream the polymerization site to be able to proceed further. As a consequence, the enzyme terminates polyadenylation after 30–40 nt and to add long poly(A) tails VP55 needs its processivity factor VP39 (30).


mRNA maturation in giant viruses: variation on a theme.

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

Mg561 and R341 are processive PolyA polymerases. (A) Time course (in minutes) PAP assay of Mg561, R341, Mg18 and a mixture of Mg18 with Mg561 or R341, on a 20-mers 5′-32P-labeled RNA primer with Mg2+ as catalytic ions. (B) As in (A), with Mn2+ instead of Mg2+ (C) Catalytic activity of Mg561 on each four nucleotides on a 20-mers and Mn2+.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402537&req=5

Figure 2: Mg561 and R341 are processive PolyA polymerases. (A) Time course (in minutes) PAP assay of Mg561, R341, Mg18 and a mixture of Mg18 with Mg561 or R341, on a 20-mers 5′-32P-labeled RNA primer with Mg2+ as catalytic ions. (B) As in (A), with Mn2+ instead of Mg2+ (C) Catalytic activity of Mg561 on each four nucleotides on a 20-mers and Mn2+.
Mentions: To confirm the functional prediction of Mg561 and R341, the proteins were purified and assayed in an in vitro polyadenylation assay on a synthetic 20-mers RNA without secondary structure in the presence of ATP and Mg2+ or Mn2+ as catalytic ions (Figure 2A and B). The two proteins were able to synthesize long products, apparently proccessively, derived from the RNA and ATP, in the presence of each cation, demonstrating the intrinsic property of Mg561 and R341 to polyadenylate RNA in absence of any specific signal (Supplementary Figure S1C, step 6). This in turn suggested that in vivo the hairpin recognition and processing could require additional proteins (Supplementary Figure S1C, step 5). In contrast to the Vaccinia and ASFV PAPs which stop after the addition of ∼30 adenylates (16,48), Mg561 and R341 were able to add much longer polyA tails (up to 700 adenylates). The nucleotide specificity of the PAPs was then assayed in the presence of Mn2+ and the 4 NTPs (Figure 2C). As expected, ATP was by far the most efficient nucleotide for homopolymeric tail synthesis. Similar results were obtained when using Mg2+ instead of Mn2+ (data not shown). However, as observed for non-canonical cellular PAPs known as terminal uridyltransferases (TUTase) or polyU polymerases (PUP) (49), Mg561 was also able to add long polyU tails, although with a reduced activity. This property was also reported for Vaccinia VP55 (29). This enzyme requires uridylates 30–40 nt upstream the polymerization site to be able to proceed further. As a consequence, the enzyme terminates polyadenylation after 30–40 nt and to add long poly(A) tails VP55 needs its processivity factor VP39 (30).

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