Limits...
AID can restrict L1 retrotransposition suggesting a dual role in innate and adaptive immunity.

MacDuff DA, Demorest ZL, Harris RS - Nucleic Acids Res. (2009)

Bottom Line: We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism.This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally.Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
Retrotransposons make up over 40% of the mammalian genome. Some copies are still capable of mobilizing and new insertions promote genetic variation. Several members of the APOBEC3 family of DNA cytosine deaminases function to limit the replication of a variety of retroelements, such as the long-terminal repeat (LTR)-containing MusD and Ty1 elements, and that of the non-LTR retrotransposons, L1 and Alu. However, the APOBEC3 genes are limited to mammalian lineages, whereas retrotransposons are far more widespread. This raises the question of what cellular factors control retroelement transposition in species that lack APOBEC3 genes. A strong phylogenetic case can be made that an ancestral activation-induced deaminase (AID)-like gene duplicated and diverged to root the APOBEC3 lineage in mammals. Therefore, we tested the hypothesis that present-day AID proteins possess anti-retroelement activity. We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism. This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally. Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity.

Show MeSH

Related in: MedlinePlus

Inhibition of L1 retrotransposition by AID and A3 proteins. (A) Percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID/A3A plasmids. Bars represent the mean of three independent transfections. Error bars represent the standard deviation. (B) Western blot showing expression of the HA-tagged AID/A3 proteins from a representative experiment from (A). Tubulin is a loading control. (C) Comparison of the percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with L1 and varying amounts of the indicted AID and A3 plasmids and their corresponding catalytically inactive mutants. The amount of AID/A3 plasmid transfected is indicated below the graph (1.0, 0.5 or 0.25 μg). The histogram bars represent the mean of three independent cultures, and the standard deviation is shown. (D) Western blot analysis of the protein expression levels from the experiment in (C). Tubulin is a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665220&req=5

Figure 2: Inhibition of L1 retrotransposition by AID and A3 proteins. (A) Percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID/A3A plasmids. Bars represent the mean of three independent transfections. Error bars represent the standard deviation. (B) Western blot showing expression of the HA-tagged AID/A3 proteins from a representative experiment from (A). Tubulin is a loading control. (C) Comparison of the percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with L1 and varying amounts of the indicted AID and A3 plasmids and their corresponding catalytically inactive mutants. The amount of AID/A3 plasmid transfected is indicated below the graph (1.0, 0.5 or 0.25 μg). The histogram bars represent the mean of three independent cultures, and the standard deviation is shown. (D) Western blot analysis of the protein expression levels from the experiment in (C). Tubulin is a loading control.

Mentions: The 293 cells were co-transfected with AID-HA expression plasmids and the L1 reporter plasmid, and transfectants were selected with puromycin (L1 plasmid). L1 retrotransposition was monitored by the appearance of GFP-positive cells 5 days later. All of the AID proteins tested inhibited L1 retrotransposition (the appearance of GFP+ cells) (Figure 2A). The ability of pig AID to inhibit L1 transposition was comparable with the positive control, human A3A [7.3-fold and 8.0-fold, respectively (23,25,31,32)]. Human AID displayed a 3.8-fold inhibition and, pufferfish AID showed a 3.2-fold inhibition despite its lower expression level (Figure 2B) and activity in E. coli (Figure 1C and Supplementary Figure S2). Chicken and zebrafish AIDs had the weakest activity against L1 at 1.7-fold and 1.3-fold, respectively. Mouse and rat AIDs showed intermediate levels of activity. The A3A catalytic site mutant, (A3AE72A) was used as a negative control (23,25,31,32). The inter-species differences may be attributable to multiple factors, including amino acid differences (Figure 1A), but it is important to emphasize that these results clearly demonstrated that the AID proteins of multiple species are capable of inhibiting L1 retrotransposition.Figure 2.


AID can restrict L1 retrotransposition suggesting a dual role in innate and adaptive immunity.

MacDuff DA, Demorest ZL, Harris RS - Nucleic Acids Res. (2009)

Inhibition of L1 retrotransposition by AID and A3 proteins. (A) Percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID/A3A plasmids. Bars represent the mean of three independent transfections. Error bars represent the standard deviation. (B) Western blot showing expression of the HA-tagged AID/A3 proteins from a representative experiment from (A). Tubulin is a loading control. (C) Comparison of the percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with L1 and varying amounts of the indicted AID and A3 plasmids and their corresponding catalytically inactive mutants. The amount of AID/A3 plasmid transfected is indicated below the graph (1.0, 0.5 or 0.25 μg). The histogram bars represent the mean of three independent cultures, and the standard deviation is shown. (D) Western blot analysis of the protein expression levels from the experiment in (C). Tubulin is a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665220&req=5

Figure 2: Inhibition of L1 retrotransposition by AID and A3 proteins. (A) Percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID/A3A plasmids. Bars represent the mean of three independent transfections. Error bars represent the standard deviation. (B) Western blot showing expression of the HA-tagged AID/A3 proteins from a representative experiment from (A). Tubulin is a loading control. (C) Comparison of the percentage of the drug-resistant cells that were GFP-positive 5 days after transfection with L1 and varying amounts of the indicted AID and A3 plasmids and their corresponding catalytically inactive mutants. The amount of AID/A3 plasmid transfected is indicated below the graph (1.0, 0.5 or 0.25 μg). The histogram bars represent the mean of three independent cultures, and the standard deviation is shown. (D) Western blot analysis of the protein expression levels from the experiment in (C). Tubulin is a loading control.
Mentions: The 293 cells were co-transfected with AID-HA expression plasmids and the L1 reporter plasmid, and transfectants were selected with puromycin (L1 plasmid). L1 retrotransposition was monitored by the appearance of GFP-positive cells 5 days later. All of the AID proteins tested inhibited L1 retrotransposition (the appearance of GFP+ cells) (Figure 2A). The ability of pig AID to inhibit L1 transposition was comparable with the positive control, human A3A [7.3-fold and 8.0-fold, respectively (23,25,31,32)]. Human AID displayed a 3.8-fold inhibition and, pufferfish AID showed a 3.2-fold inhibition despite its lower expression level (Figure 2B) and activity in E. coli (Figure 1C and Supplementary Figure S2). Chicken and zebrafish AIDs had the weakest activity against L1 at 1.7-fold and 1.3-fold, respectively. Mouse and rat AIDs showed intermediate levels of activity. The A3A catalytic site mutant, (A3AE72A) was used as a negative control (23,25,31,32). The inter-species differences may be attributable to multiple factors, including amino acid differences (Figure 1A), but it is important to emphasize that these results clearly demonstrated that the AID proteins of multiple species are capable of inhibiting L1 retrotransposition.Figure 2.

Bottom Line: We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism.This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally.Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
Retrotransposons make up over 40% of the mammalian genome. Some copies are still capable of mobilizing and new insertions promote genetic variation. Several members of the APOBEC3 family of DNA cytosine deaminases function to limit the replication of a variety of retroelements, such as the long-terminal repeat (LTR)-containing MusD and Ty1 elements, and that of the non-LTR retrotransposons, L1 and Alu. However, the APOBEC3 genes are limited to mammalian lineages, whereas retrotransposons are far more widespread. This raises the question of what cellular factors control retroelement transposition in species that lack APOBEC3 genes. A strong phylogenetic case can be made that an ancestral activation-induced deaminase (AID)-like gene duplicated and diverged to root the APOBEC3 lineage in mammals. Therefore, we tested the hypothesis that present-day AID proteins possess anti-retroelement activity. We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism. This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally. Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity.

Show MeSH
Related in: MedlinePlus