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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.

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Effect of AID catalytic and DNA-binding mutants on L1 retrotransposition. (A) Percentage of puromycin-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID variants. A3A and A3AE72A were included as controls. Histogram bars represent the mean of three independent cultures, and the standard deviation is shown. WT, wild-type. (B) Western blot showing expression of the HA-tagged proteins from a representative experiment from (A). Tubulin is a loading control. (C) E. coli-based RifR mutation assay. Each ‘x’ represents the mutation frequency of an independent culture, calculated as the number of rifampicin-resistant colonies per viable cell. Eight independent cultures were assayed for each AID variant, and the median mutation frequencies are indicated by the horizontal bars. Vector represents the background level of mutation.
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Figure 3: Effect of AID catalytic and DNA-binding mutants on L1 retrotransposition. (A) Percentage of puromycin-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID variants. A3A and A3AE72A were included as controls. Histogram bars represent the mean of three independent cultures, and the standard deviation is shown. WT, wild-type. (B) Western blot showing expression of the HA-tagged proteins from a representative experiment from (A). Tubulin is a loading control. (C) E. coli-based RifR mutation assay. Each ‘x’ represents the mutation frequency of an independent culture, calculated as the number of rifampicin-resistant colonies per viable cell. Eight independent cultures were assayed for each AID variant, and the median mutation frequencies are indicated by the horizontal bars. Vector represents the background level of mutation.

Mentions: To confirm and extend these studies, we tested several mutants of human AID that were expected to disrupt catalysis by preventing zinc coordination (C87A, C90A) or altering the catalytic site pocket [W84A; extrapolated from the NMR structure of the C-terminal domain of A3G (60)]. These mutants also displayed close to wild-type levels of activity against L1, despite being catalytically inactive in the E. coli-based DNA mutation assay (Figure 3 and Supplementary Figure S2). In contrast, consistent with the requirement of the integrity of the catalytic site of A3A for L1 restriction, mutation of W98 (equivalent to W84 in AID) to alanine completely eliminated the anti-L1 activity of A3A (Figure 3). These data indicated that AID and A3A restrict L1 replication by two distinct mechanisms.Figure 3.


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

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

Effect of AID catalytic and DNA-binding mutants on L1 retrotransposition. (A) Percentage of puromycin-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID variants. A3A and A3AE72A were included as controls. Histogram bars represent the mean of three independent cultures, and the standard deviation is shown. WT, wild-type. (B) Western blot showing expression of the HA-tagged proteins from a representative experiment from (A). Tubulin is a loading control. (C) E. coli-based RifR mutation assay. Each ‘x’ represents the mutation frequency of an independent culture, calculated as the number of rifampicin-resistant colonies per viable cell. Eight independent cultures were assayed for each AID variant, and the median mutation frequencies are indicated by the horizontal bars. Vector represents the background level of mutation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Effect of AID catalytic and DNA-binding mutants on L1 retrotransposition. (A) Percentage of puromycin-resistant cells that were GFP-positive 5 days after transfection with the L1 and indicated AID variants. A3A and A3AE72A were included as controls. Histogram bars represent the mean of three independent cultures, and the standard deviation is shown. WT, wild-type. (B) Western blot showing expression of the HA-tagged proteins from a representative experiment from (A). Tubulin is a loading control. (C) E. coli-based RifR mutation assay. Each ‘x’ represents the mutation frequency of an independent culture, calculated as the number of rifampicin-resistant colonies per viable cell. Eight independent cultures were assayed for each AID variant, and the median mutation frequencies are indicated by the horizontal bars. Vector represents the background level of mutation.
Mentions: To confirm and extend these studies, we tested several mutants of human AID that were expected to disrupt catalysis by preventing zinc coordination (C87A, C90A) or altering the catalytic site pocket [W84A; extrapolated from the NMR structure of the C-terminal domain of A3G (60)]. These mutants also displayed close to wild-type levels of activity against L1, despite being catalytically inactive in the E. coli-based DNA mutation assay (Figure 3 and Supplementary Figure S2). In contrast, consistent with the requirement of the integrity of the catalytic site of A3A for L1 restriction, mutation of W98 (equivalent to W84 in AID) to alanine completely eliminated the anti-L1 activity of A3A (Figure 3). These data indicated that AID and A3A restrict L1 replication by two distinct mechanisms.Figure 3.

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