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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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Viral AlkB proteins used in the present study. A sequence alignment was generated from 23 viral AlkB protein sequences, and the five sequences shown were extracted from this alignment. The shading pattern of this 23 sequence alignment has been maintained (explaining why some residues are shaded without being conserved among the five sequences shown). Arrows indicate the borders of the AlkB-coding regions included in the various expression constructs. The solid line indicates the ‘AlkB core’, i.e. the region which displays homology to other members of the AlkB family, and extensive sequence homology within the subfamily of viral AlkB proteins. The dotted line indicates a region where sequence conservation is observed within the group of 23 viral AlkB sequences (but not shared by other AlkB family members): (1) CXC in 14 sequences; (2) I/L/V/M-X-I/L/V in 15 sequences; 12 sequences adhered to the consensus CXCX3-I/L/V/M-X-I/L/V.
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Figure 3: Viral AlkB proteins used in the present study. A sequence alignment was generated from 23 viral AlkB protein sequences, and the five sequences shown were extracted from this alignment. The shading pattern of this 23 sequence alignment has been maintained (explaining why some residues are shaded without being conserved among the five sequences shown). Arrows indicate the borders of the AlkB-coding regions included in the various expression constructs. The solid line indicates the ‘AlkB core’, i.e. the region which displays homology to other members of the AlkB family, and extensive sequence homology within the subfamily of viral AlkB proteins. The dotted line indicates a region where sequence conservation is observed within the group of 23 viral AlkB sequences (but not shared by other AlkB family members): (1) CXC in 14 sequences; (2) I/L/V/M-X-I/L/V in 15 sequences; 12 sequences adhered to the consensus CXCX3-I/L/V/M-X-I/L/V.

Mentions: AlkB proteins from five plant viruses, i.e. GVA, BlScV, BVY, Citrus leaf blotch virus (CLBV) and Little cherry virus 2 (LChV-2) were selected for initial functional characterization. These viruses represent three divergent families, Flexiviridae (GVA, BlScV and CLBV), Closteroviridae (LChV-2) and Potyviridae (BVY). The viral AlkB domain is part of a large replicase polyprotein, where it is flanked by long stretches of low sequence conservation. It is therefore not trivial to define the N- and C-terminal borders of the functionally active viral AlkB proteins. However, based on sequence homology within the viral AlkB family, as well as sequence comparison with bacterial AlkBs, a viral AlkB core region could be defined (Figure 3). This core region of ∼132 aa is substantially smaller than a typical AlkB protein from bacteria (190–220 aa), suggesting that parts of the less-conserved regions flanking the viral AlkB core may be required to form a functional AlkB protein. Indeed, some sequence conservation within the viral AlkB group, not shared with other AlkB proteins, is observed in a region of 20–25 aa upstream of the viral AlkB core (Figure 3). Based on this observation, viral AlkB proteins with N-terminal extensions of different length were constructed (Figure 3). The border of these extensions was chosen rather arbitrarily, but without disrupting predicted secondary structure elements. This resulted in viral AlkB proteins with N-terminal extensions of 0, 36, 50 or 94 aa, designated accordingly (GVA-36, BlScV-94, etc.). The C-terminal border of the viral AlkB proteins was set at 6 aa downstream of the characteristic RX5R motif (Figures 3, S1 and S3), which is found close to the C-terminus of most AlkB proteins, and is usually followed by 5–20 non-conserved residues.Figure 3.


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)

Viral AlkB proteins used in the present study. A sequence alignment was generated from 23 viral AlkB protein sequences, and the five sequences shown were extracted from this alignment. The shading pattern of this 23 sequence alignment has been maintained (explaining why some residues are shaded without being conserved among the five sequences shown). Arrows indicate the borders of the AlkB-coding regions included in the various expression constructs. The solid line indicates the ‘AlkB core’, i.e. the region which displays homology to other members of the AlkB family, and extensive sequence homology within the subfamily of viral AlkB proteins. The dotted line indicates a region where sequence conservation is observed within the group of 23 viral AlkB sequences (but not shared by other AlkB family members): (1) CXC in 14 sequences; (2) I/L/V/M-X-I/L/V in 15 sequences; 12 sequences adhered to the consensus CXCX3-I/L/V/M-X-I/L/V.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553587&req=5

Figure 3: Viral AlkB proteins used in the present study. A sequence alignment was generated from 23 viral AlkB protein sequences, and the five sequences shown were extracted from this alignment. The shading pattern of this 23 sequence alignment has been maintained (explaining why some residues are shaded without being conserved among the five sequences shown). Arrows indicate the borders of the AlkB-coding regions included in the various expression constructs. The solid line indicates the ‘AlkB core’, i.e. the region which displays homology to other members of the AlkB family, and extensive sequence homology within the subfamily of viral AlkB proteins. The dotted line indicates a region where sequence conservation is observed within the group of 23 viral AlkB sequences (but not shared by other AlkB family members): (1) CXC in 14 sequences; (2) I/L/V/M-X-I/L/V in 15 sequences; 12 sequences adhered to the consensus CXCX3-I/L/V/M-X-I/L/V.
Mentions: AlkB proteins from five plant viruses, i.e. GVA, BlScV, BVY, Citrus leaf blotch virus (CLBV) and Little cherry virus 2 (LChV-2) were selected for initial functional characterization. These viruses represent three divergent families, Flexiviridae (GVA, BlScV and CLBV), Closteroviridae (LChV-2) and Potyviridae (BVY). The viral AlkB domain is part of a large replicase polyprotein, where it is flanked by long stretches of low sequence conservation. It is therefore not trivial to define the N- and C-terminal borders of the functionally active viral AlkB proteins. However, based on sequence homology within the viral AlkB family, as well as sequence comparison with bacterial AlkBs, a viral AlkB core region could be defined (Figure 3). This core region of ∼132 aa is substantially smaller than a typical AlkB protein from bacteria (190–220 aa), suggesting that parts of the less-conserved regions flanking the viral AlkB core may be required to form a functional AlkB protein. Indeed, some sequence conservation within the viral AlkB group, not shared with other AlkB proteins, is observed in a region of 20–25 aa upstream of the viral AlkB core (Figure 3). Based on this observation, viral AlkB proteins with N-terminal extensions of different length were constructed (Figure 3). The border of these extensions was chosen rather arbitrarily, but without disrupting predicted secondary structure elements. This resulted in viral AlkB proteins with N-terminal extensions of 0, 36, 50 or 94 aa, designated accordingly (GVA-36, BlScV-94, etc.). The C-terminal border of the viral AlkB proteins was set at 6 aa downstream of the characteristic RX5R motif (Figures 3, S1 and S3), which is found close to the C-terminus of most AlkB proteins, and is usually followed by 5–20 non-conserved residues.Figure 3.

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