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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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POLA1 deficiency is associated with reduced cytosolic RNA:DNA levels(a) Immunoprecipitation of dsDNA and RNA:DNA from cytosolic fractions of HEK293T cells using the corresponding antibodies and Protein-G agarose beads. Beads were stained with Picogreen® and visualized by fluorescence microscopy. Nucleic acid recovery was quantified by image analysis (ImageJ). Mean relative fluorescence levels are indicated in parantheses. (b) As in (a), dsDNA or RNA:DNA were quantified in control or XLPDR-derived fibroblasts. (c) RNA:DNA was quantified in a similar manner in three models of POLA1 deficiency: HEK293T and HeLa after POLA1 silencing (compared to control siRNA), and XLPDR-derived dermal fibroblasts (compared to control cells). Representative images are shown and relative fluorescence levels are indicated. (d) Cytosolic RNA:DNA content was quantified in XLPDR-derived fibroblasts stably transduced with a lentivirus encoding HA-tagged wild-type POLA1 (HA-POLA1wt) or EV. Representative images and relative fluorescence levels are indicated. (e) Immunoblot analysis of total and p-TBK1, and total and HA-tagged POLA1 expression in XLPDR fibroblast stably expressing HA-POLA1wt or EV (as in d). (f) qRT-PCR analysis of IFIT1 mRNA expression in the same cells depicted in (d); when indicated, the cells were treated with poly(dA:dT) for 16h. Scale bars are 20 μm. *p≤0.05, **p≤0.01, NS – not significant (unpaired Student’s t-test). In (f) all comparisons are made against the corresponding WT conditions. Data are representative of 1 (d,e), 2 (a,b,f), or 4 (c) independent experiments (mean (a,c,d) and mean and s.e.m.(b,f)).
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Figure 5: POLA1 deficiency is associated with reduced cytosolic RNA:DNA levels(a) Immunoprecipitation of dsDNA and RNA:DNA from cytosolic fractions of HEK293T cells using the corresponding antibodies and Protein-G agarose beads. Beads were stained with Picogreen® and visualized by fluorescence microscopy. Nucleic acid recovery was quantified by image analysis (ImageJ). Mean relative fluorescence levels are indicated in parantheses. (b) As in (a), dsDNA or RNA:DNA were quantified in control or XLPDR-derived fibroblasts. (c) RNA:DNA was quantified in a similar manner in three models of POLA1 deficiency: HEK293T and HeLa after POLA1 silencing (compared to control siRNA), and XLPDR-derived dermal fibroblasts (compared to control cells). Representative images are shown and relative fluorescence levels are indicated. (d) Cytosolic RNA:DNA content was quantified in XLPDR-derived fibroblasts stably transduced with a lentivirus encoding HA-tagged wild-type POLA1 (HA-POLA1wt) or EV. Representative images and relative fluorescence levels are indicated. (e) Immunoblot analysis of total and p-TBK1, and total and HA-tagged POLA1 expression in XLPDR fibroblast stably expressing HA-POLA1wt or EV (as in d). (f) qRT-PCR analysis of IFIT1 mRNA expression in the same cells depicted in (d); when indicated, the cells were treated with poly(dA:dT) for 16h. Scale bars are 20 μm. *p≤0.05, **p≤0.01, NS – not significant (unpaired Student’s t-test). In (f) all comparisons are made against the corresponding WT conditions. Data are representative of 1 (d,e), 2 (a,b,f), or 4 (c) independent experiments (mean (a,c,d) and mean and s.e.m.(b,f)).

Mentions: To explain the constitutive activation of the IRF pathway in XLPDR, we examined whether increased amounts of cytosolic nucleic acids could be a possible trigger. To this end, we used antibodies with defined reactivities against specific nucleic acids to immunoprecipitate these molecules from cytosolic fractions and stained the recovered material with PicoGreen®, a double-stranded nucleic acid dye (Fig. 5a). Cytosolic dsDNA was mildly decreased in XLPDR fibroblasts, whereas cytosolic RNA:DNA was almost undetectable (Fig. 5b). The reduction in cytosolic RNA:DNA observed in XLPDR fibroblasts was also seen after silencing of POLA1 in HEK293T and HeLa cells (Fig. 5c and Supplementary Fig. 6a,b).


DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis
POLA1 deficiency is associated with reduced cytosolic RNA:DNA levels(a) Immunoprecipitation of dsDNA and RNA:DNA from cytosolic fractions of HEK293T cells using the corresponding antibodies and Protein-G agarose beads. Beads were stained with Picogreen® and visualized by fluorescence microscopy. Nucleic acid recovery was quantified by image analysis (ImageJ). Mean relative fluorescence levels are indicated in parantheses. (b) As in (a), dsDNA or RNA:DNA were quantified in control or XLPDR-derived fibroblasts. (c) RNA:DNA was quantified in a similar manner in three models of POLA1 deficiency: HEK293T and HeLa after POLA1 silencing (compared to control siRNA), and XLPDR-derived dermal fibroblasts (compared to control cells). Representative images are shown and relative fluorescence levels are indicated. (d) Cytosolic RNA:DNA content was quantified in XLPDR-derived fibroblasts stably transduced with a lentivirus encoding HA-tagged wild-type POLA1 (HA-POLA1wt) or EV. Representative images and relative fluorescence levels are indicated. (e) Immunoblot analysis of total and p-TBK1, and total and HA-tagged POLA1 expression in XLPDR fibroblast stably expressing HA-POLA1wt or EV (as in d). (f) qRT-PCR analysis of IFIT1 mRNA expression in the same cells depicted in (d); when indicated, the cells were treated with poly(dA:dT) for 16h. Scale bars are 20 μm. *p≤0.05, **p≤0.01, NS – not significant (unpaired Student’s t-test). In (f) all comparisons are made against the corresponding WT conditions. Data are representative of 1 (d,e), 2 (a,b,f), or 4 (c) independent experiments (mean (a,c,d) and mean and s.e.m.(b,f)).
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Figure 5: POLA1 deficiency is associated with reduced cytosolic RNA:DNA levels(a) Immunoprecipitation of dsDNA and RNA:DNA from cytosolic fractions of HEK293T cells using the corresponding antibodies and Protein-G agarose beads. Beads were stained with Picogreen® and visualized by fluorescence microscopy. Nucleic acid recovery was quantified by image analysis (ImageJ). Mean relative fluorescence levels are indicated in parantheses. (b) As in (a), dsDNA or RNA:DNA were quantified in control or XLPDR-derived fibroblasts. (c) RNA:DNA was quantified in a similar manner in three models of POLA1 deficiency: HEK293T and HeLa after POLA1 silencing (compared to control siRNA), and XLPDR-derived dermal fibroblasts (compared to control cells). Representative images are shown and relative fluorescence levels are indicated. (d) Cytosolic RNA:DNA content was quantified in XLPDR-derived fibroblasts stably transduced with a lentivirus encoding HA-tagged wild-type POLA1 (HA-POLA1wt) or EV. Representative images and relative fluorescence levels are indicated. (e) Immunoblot analysis of total and p-TBK1, and total and HA-tagged POLA1 expression in XLPDR fibroblast stably expressing HA-POLA1wt or EV (as in d). (f) qRT-PCR analysis of IFIT1 mRNA expression in the same cells depicted in (d); when indicated, the cells were treated with poly(dA:dT) for 16h. Scale bars are 20 μm. *p≤0.05, **p≤0.01, NS – not significant (unpaired Student’s t-test). In (f) all comparisons are made against the corresponding WT conditions. Data are representative of 1 (d,e), 2 (a,b,f), or 4 (c) independent experiments (mean (a,c,d) and mean and s.e.m.(b,f)).
Mentions: To explain the constitutive activation of the IRF pathway in XLPDR, we examined whether increased amounts of cytosolic nucleic acids could be a possible trigger. To this end, we used antibodies with defined reactivities against specific nucleic acids to immunoprecipitate these molecules from cytosolic fractions and stained the recovered material with PicoGreen®, a double-stranded nucleic acid dye (Fig. 5a). Cytosolic dsDNA was mildly decreased in XLPDR fibroblasts, whereas cytosolic RNA:DNA was almost undetectable (Fig. 5b). The reduction in cytosolic RNA:DNA observed in XLPDR fibroblasts was also seen after silencing of POLA1 in HEK293T and HeLa cells (Fig. 5c and Supplementary Fig. 6a,b).

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.

No MeSH data available.


Related in: MedlinePlus