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DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response.

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Cytosolic RNA:DNA attenuates IRF activation(a) Immunoprecipitation of the indicated proteins from the cytosolic fraction of HEK293T was followed by Picogreen® staining and fluorescence quantification. Represetative images and fluorescence intensities (in parantheses) are presented (left panels); immunoblotting for the precipitated proteins is also shown (right panels). (b) POLA1 was immunoprecipitated as in (a), and the recovered beads were treated with RNaseA or DNAseI prior to Picogreen® staining. Fluorescence levels are indicated in parentheses. (c) Cytosolic POLA1 immunoprecipitation (left) was followed by nucleic acid elution and re-immunoprecipitated by RNA:DNA antibody (right). Beads were stained with Picogreen® and visualized by confocal microscopy. Relative fluorescence levels are indicated in parantheses. (d) Nucleic acids immunoprecipitated from cytosolic fractions, either directly by an RNA:DNA antibody or using a POLA1 antibody, were eluted as in (c) and analyzed by on-chip DNA or RNA capillary electrophoresis. (e) Similar to (d), nucleic acids were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (f) Cytosolic RNA:DNA content was quantified by immunoprecipitation and bead staining in HEK293T cells transfected as indicated. Representative images are shown (left) and fluorescence values are plotted (right). (g) qRT-PCR analysis of IFIT1 and IFIT2 mRNA expression in HeLa cells transfected with the indicated siRNA and also transfected with native cytosolic RNA:DNA extracted from HEK293T cells. (h) Similar to (g), but using synthetic RNA:DNA duplexes that were transfected into HeLa cells; when indicated, cells were stimulated with poly(I:C) (for 8h). Scale bars are 20 μm. *p<0.05 (unpaired Student’s t test). In (f) all values were compared to control (siCtrl, EV) conditions. In (g,h), the siPOLA1 values were compared to the corresponding conditions in the siCtrl group. Data are from 1 experiment (c), or representative of 2 (b,f,e), 3 (h) or 4 (a,g) independent experiments (mean (a) and mean and s.e.m.(f,g,h)).
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Figure 6: Cytosolic RNA:DNA attenuates IRF activation(a) Immunoprecipitation of the indicated proteins from the cytosolic fraction of HEK293T was followed by Picogreen® staining and fluorescence quantification. Represetative images and fluorescence intensities (in parantheses) are presented (left panels); immunoblotting for the precipitated proteins is also shown (right panels). (b) POLA1 was immunoprecipitated as in (a), and the recovered beads were treated with RNaseA or DNAseI prior to Picogreen® staining. Fluorescence levels are indicated in parentheses. (c) Cytosolic POLA1 immunoprecipitation (left) was followed by nucleic acid elution and re-immunoprecipitated by RNA:DNA antibody (right). Beads were stained with Picogreen® and visualized by confocal microscopy. Relative fluorescence levels are indicated in parantheses. (d) Nucleic acids immunoprecipitated from cytosolic fractions, either directly by an RNA:DNA antibody or using a POLA1 antibody, were eluted as in (c) and analyzed by on-chip DNA or RNA capillary electrophoresis. (e) Similar to (d), nucleic acids were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (f) Cytosolic RNA:DNA content was quantified by immunoprecipitation and bead staining in HEK293T cells transfected as indicated. Representative images are shown (left) and fluorescence values are plotted (right). (g) qRT-PCR analysis of IFIT1 and IFIT2 mRNA expression in HeLa cells transfected with the indicated siRNA and also transfected with native cytosolic RNA:DNA extracted from HEK293T cells. (h) Similar to (g), but using synthetic RNA:DNA duplexes that were transfected into HeLa cells; when indicated, cells were stimulated with poly(I:C) (for 8h). Scale bars are 20 μm. *p<0.05 (unpaired Student’s t test). In (f) all values were compared to control (siCtrl, EV) conditions. In (g,h), the siPOLA1 values were compared to the corresponding conditions in the siCtrl group. Data are from 1 experiment (c), or representative of 2 (b,f,e), 3 (h) or 4 (a,g) independent experiments (mean (a) and mean and s.e.m.(f,g,h)).

Mentions: As mentioned previously, the role of the Polα-Primase complex during DNA replication is to synthesize an RNA:DNA primer on a DNA template strand22, suggesting a possible direct link between POLA1 polymerase activity and the synthesis of cytosolic RNA:DNA. To test this notion, we first examined the subcellular localization of POLA1 and found that a substantial proportion of POLA1 localized to the cytosol, as determined by both immmunofluoresce staining and biochemical fractionation (Supplementary Fig. 7a,b). Moreover, cytosolic POLA1 colocalized with RNA:DNA with a speckled pattern (Supplementary Fig. 7c). Indeed, cytosolic POLA1 (and not other DNA binding proteins tested) co-immunoprecipitated with nucleic acids, as determined by PicoGreen® staining of beads (Fig. 6a). This material was sensitive to both DNase and RNase treatment (Fig. 6b), and after elution from POLA1, could in part be re-immunoprecipitated by the RNA:DNA antibody (Fig. 6c). The eluted nucleic acids could be resolved by on-chip capillary electrophoresis or by agarose gel electrophoresis and consisted predominantly of 40–100 bp fragments (Fig. 6d,e). Thus, cytosolic POLA1 associates with short nucleic acids, which at least in part include RNA:DNA.


DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis
Cytosolic RNA:DNA attenuates IRF activation(a) Immunoprecipitation of the indicated proteins from the cytosolic fraction of HEK293T was followed by Picogreen® staining and fluorescence quantification. Represetative images and fluorescence intensities (in parantheses) are presented (left panels); immunoblotting for the precipitated proteins is also shown (right panels). (b) POLA1 was immunoprecipitated as in (a), and the recovered beads were treated with RNaseA or DNAseI prior to Picogreen® staining. Fluorescence levels are indicated in parentheses. (c) Cytosolic POLA1 immunoprecipitation (left) was followed by nucleic acid elution and re-immunoprecipitated by RNA:DNA antibody (right). Beads were stained with Picogreen® and visualized by confocal microscopy. Relative fluorescence levels are indicated in parantheses. (d) Nucleic acids immunoprecipitated from cytosolic fractions, either directly by an RNA:DNA antibody or using a POLA1 antibody, were eluted as in (c) and analyzed by on-chip DNA or RNA capillary electrophoresis. (e) Similar to (d), nucleic acids were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (f) Cytosolic RNA:DNA content was quantified by immunoprecipitation and bead staining in HEK293T cells transfected as indicated. Representative images are shown (left) and fluorescence values are plotted (right). (g) qRT-PCR analysis of IFIT1 and IFIT2 mRNA expression in HeLa cells transfected with the indicated siRNA and also transfected with native cytosolic RNA:DNA extracted from HEK293T cells. (h) Similar to (g), but using synthetic RNA:DNA duplexes that were transfected into HeLa cells; when indicated, cells were stimulated with poly(I:C) (for 8h). Scale bars are 20 μm. *p<0.05 (unpaired Student’s t test). In (f) all values were compared to control (siCtrl, EV) conditions. In (g,h), the siPOLA1 values were compared to the corresponding conditions in the siCtrl group. Data are from 1 experiment (c), or representative of 2 (b,f,e), 3 (h) or 4 (a,g) independent experiments (mean (a) and mean and s.e.m.(f,g,h)).
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Figure 6: Cytosolic RNA:DNA attenuates IRF activation(a) Immunoprecipitation of the indicated proteins from the cytosolic fraction of HEK293T was followed by Picogreen® staining and fluorescence quantification. Represetative images and fluorescence intensities (in parantheses) are presented (left panels); immunoblotting for the precipitated proteins is also shown (right panels). (b) POLA1 was immunoprecipitated as in (a), and the recovered beads were treated with RNaseA or DNAseI prior to Picogreen® staining. Fluorescence levels are indicated in parentheses. (c) Cytosolic POLA1 immunoprecipitation (left) was followed by nucleic acid elution and re-immunoprecipitated by RNA:DNA antibody (right). Beads were stained with Picogreen® and visualized by confocal microscopy. Relative fluorescence levels are indicated in parantheses. (d) Nucleic acids immunoprecipitated from cytosolic fractions, either directly by an RNA:DNA antibody or using a POLA1 antibody, were eluted as in (c) and analyzed by on-chip DNA or RNA capillary electrophoresis. (e) Similar to (d), nucleic acids were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (f) Cytosolic RNA:DNA content was quantified by immunoprecipitation and bead staining in HEK293T cells transfected as indicated. Representative images are shown (left) and fluorescence values are plotted (right). (g) qRT-PCR analysis of IFIT1 and IFIT2 mRNA expression in HeLa cells transfected with the indicated siRNA and also transfected with native cytosolic RNA:DNA extracted from HEK293T cells. (h) Similar to (g), but using synthetic RNA:DNA duplexes that were transfected into HeLa cells; when indicated, cells were stimulated with poly(I:C) (for 8h). Scale bars are 20 μm. *p<0.05 (unpaired Student’s t test). In (f) all values were compared to control (siCtrl, EV) conditions. In (g,h), the siPOLA1 values were compared to the corresponding conditions in the siCtrl group. Data are from 1 experiment (c), or representative of 2 (b,f,e), 3 (h) or 4 (a,g) independent experiments (mean (a) and mean and s.e.m.(f,g,h)).
Mentions: As mentioned previously, the role of the Polα-Primase complex during DNA replication is to synthesize an RNA:DNA primer on a DNA template strand22, suggesting a possible direct link between POLA1 polymerase activity and the synthesis of cytosolic RNA:DNA. To test this notion, we first examined the subcellular localization of POLA1 and found that a substantial proportion of POLA1 localized to the cytosol, as determined by both immmunofluoresce staining and biochemical fractionation (Supplementary Fig. 7a,b). Moreover, cytosolic POLA1 colocalized with RNA:DNA with a speckled pattern (Supplementary Fig. 7c). Indeed, cytosolic POLA1 (and not other DNA binding proteins tested) co-immunoprecipitated with nucleic acids, as determined by PicoGreen® staining of beads (Fig. 6a). This material was sensitive to both DNase and RNase treatment (Fig. 6b), and after elution from POLA1, could in part be re-immunoprecipitated by the RNA:DNA antibody (Fig. 6c). The eluted nucleic acids could be resolved by on-chip capillary electrophoresis or by agarose gel electrophoresis and consisted predominantly of 40–100 bp fragments (Fig. 6d,e). Thus, cytosolic POLA1 associates with short nucleic acids, which at least in part include RNA:DNA.

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-&alpha;. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response.

No MeSH data available.


Related in: MedlinePlus