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Viral AlkB proteins repair RNA damage by oxidative demethylation.

van den Born E, Omelchenko MV, Bekkelund A, Leihne V, Koonin EV, Dolja VV, Falnes PØ - Nucleic Acids Res. (2008)

Bottom Line: The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro.Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity.Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

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Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.
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Figure 1: Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.

Mentions: In an attempt to gain insight into the origin and evolution of the AlkB homologues encoded in genomes of plant viruses, a two-tier phylogenetic analysis of the AlkB family was performed. First, a large alignment was constructed that included representative sets of AlkB proteins from bacteria, eukaryotes and viruses (Figure S1), and a phylogenetic tree was built, aiming at deciphering the ultimate origin of the viral AlkBs. In the resulting tree, the viral sequences formed one of the three major, well-separated branches that is strongly supported by bootstrap analysis, whereas the remaining two branches both included assortments of bacterial and eukaryotic members of the family (Figures 1A and S2). Because no independent information was available to root this tree, the origin of viral AlkBs could formally not be inferred. However, viral AlkBs were significantly more similar to bacterial homologues than to eukaryotic ones. This was also reflected by the fact that the first iteration of BLASTp searches with viral AlkB sequences yielded statistically significant hits (E-values in the range of 10−5 to 10−4) to many bacterial AlkBs, especially those from cyanobacteria, but not to eukaryotic AlkBs. Notably, the viral AlkB clade in the family tree is well separated from the plant homologues (Figure 1A).


Viral AlkB proteins repair RNA damage by oxidative demethylation.

van den Born E, Omelchenko MV, Bekkelund A, Leihne V, Koonin EV, Dolja VV, Falnes PØ - Nucleic Acids Res. (2008)

Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553587&req=5

Figure 1: Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.
Mentions: In an attempt to gain insight into the origin and evolution of the AlkB homologues encoded in genomes of plant viruses, a two-tier phylogenetic analysis of the AlkB family was performed. First, a large alignment was constructed that included representative sets of AlkB proteins from bacteria, eukaryotes and viruses (Figure S1), and a phylogenetic tree was built, aiming at deciphering the ultimate origin of the viral AlkBs. In the resulting tree, the viral sequences formed one of the three major, well-separated branches that is strongly supported by bootstrap analysis, whereas the remaining two branches both included assortments of bacterial and eukaryotic members of the family (Figures 1A and S2). Because no independent information was available to root this tree, the origin of viral AlkBs could formally not be inferred. However, viral AlkBs were significantly more similar to bacterial homologues than to eukaryotic ones. This was also reflected by the fact that the first iteration of BLASTp searches with viral AlkB sequences yielded statistically significant hits (E-values in the range of 10−5 to 10−4) to many bacterial AlkBs, especially those from cyanobacteria, but not to eukaryotic AlkBs. Notably, the viral AlkB clade in the family tree is well separated from the plant homologues (Figure 1A).

Bottom Line: The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro.Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity.Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

Show MeSH
Related in: MedlinePlus