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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.

No MeSH data available.


XLPDR is due to an intronic mutation that disrupts POLA1 expression(a) RNA-seq quantification of gene expression across the XLPDR linkage interval in XLPDR-derived dermal fibroblasts (P1, P2, P3) was normalized against four normal fibroblast cell lines (F1, and three lines from unrelated males, U1, U2, U3). Only genes with detectable expression are displayed. (b) Immunoblotting determination of POLA1 protein expression in dermal fibroblasts from unaffected control lines and XLPDR-derived cells. (c) Quantitative RT-PCR (qRT-PCR) for POLA1 mRNA (left panel) in multiple independent samples from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines). POLA1 mRNA level is normalized to ACTB (×10−3). In addition, quantification of POLA1 protein levels shown in (b) are similarly presented (right panel). (d) Schematic representation of the mutation in intron 13 of the POLA1 gene. The location of primers used for RT-PCR are noted. Ex – exon. (e) RT-PCR using primers A and B (see d) and RNA from XLPDR-derived fibroblasts and a control line. The amplified products were separated by agarose gel electrophoresis and visualized with ethidium bromide. (f) qRT-PCR using transcript-specific primers for wild-type and misspliced forms of POLA1 mRNA from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines) was performed. (g) Exon trapping analysis in cells transfected with pSpliceExpress plasmids containing empty vector (EV), the wild type POLA1 intron 13 (A), or the intron bearing the XLPDR mutation (A→G). RT-PCR amplified products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining. *p<0.005, **p<0.001 (unpaired Student’s t-test). Data are pooled from 3 independent experiments (a), or representative from 2 (e,f,g) or 4 (b,e) independent experiments (mean and s.e.m.(a) or mean and individual sample values (c,f)).
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Figure 2: XLPDR is due to an intronic mutation that disrupts POLA1 expression(a) RNA-seq quantification of gene expression across the XLPDR linkage interval in XLPDR-derived dermal fibroblasts (P1, P2, P3) was normalized against four normal fibroblast cell lines (F1, and three lines from unrelated males, U1, U2, U3). Only genes with detectable expression are displayed. (b) Immunoblotting determination of POLA1 protein expression in dermal fibroblasts from unaffected control lines and XLPDR-derived cells. (c) Quantitative RT-PCR (qRT-PCR) for POLA1 mRNA (left panel) in multiple independent samples from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines). POLA1 mRNA level is normalized to ACTB (×10−3). In addition, quantification of POLA1 protein levels shown in (b) are similarly presented (right panel). (d) Schematic representation of the mutation in intron 13 of the POLA1 gene. The location of primers used for RT-PCR are noted. Ex – exon. (e) RT-PCR using primers A and B (see d) and RNA from XLPDR-derived fibroblasts and a control line. The amplified products were separated by agarose gel electrophoresis and visualized with ethidium bromide. (f) qRT-PCR using transcript-specific primers for wild-type and misspliced forms of POLA1 mRNA from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines) was performed. (g) Exon trapping analysis in cells transfected with pSpliceExpress plasmids containing empty vector (EV), the wild type POLA1 intron 13 (A), or the intron bearing the XLPDR mutation (A→G). RT-PCR amplified products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining. *p<0.005, **p<0.001 (unpaired Student’s t-test). Data are pooled from 3 independent experiments (a), or representative from 2 (e,f,g) or 4 (b,e) independent experiments (mean and s.e.m.(a) or mean and individual sample values (c,f)).

Mentions: Next, using dermal fibroblasts derived from three XLPDR probands and five control lines, we compared the expression levels of genes from the XLPDR linkage interval. Only POLA1 was significantly underexpressed in XLPDR cells (Fig. 2a). Furthermore, POLA1 protein expression in fibroblasts was markedly reduced as well (Fig. 2b). Quantitation using Li-COR indicated that POLA1 protein levels in XLPDR fibroblasts were reduced to about 35% of that seen in wild-type cells, and this was closely matched by the reduction observed by qRT-PCR at the mRNA level (Fig. 2c). An analogous effect on mRNA and protein expression was noted in patient-derived lymphoblastoid cell lines (LCLs, Supplementary Fig. 1d,e,f). Despite having reduced POLA1 levels, mutant fibroblasts did not display altered proliferative capacity or changes in cell cycle distribution (Supplementary Fig. 2a,b). Similarly, mitogen-induced lymphocyte proliferation was unaffected in two probands from the Waco, Texas family (Supplementary Fig. 2c). Furthermore, POLA1 silencing by siRNA to a degree comparable to that seen in XLPDR did not lead to cell cycle arrest in HeLa cells with an integrated cell-cycle reporter system (Supplementary Fig. 2d). Thus, the reduction of POLA1 expression in XLPDR did not appear to compromise cell replication, in agreement with the lack of a growth phenotype in most patients.


DNA polymerase- α regulates type I interferon activation through cytosolic RNA:DNA synthesis
XLPDR is due to an intronic mutation that disrupts POLA1 expression(a) RNA-seq quantification of gene expression across the XLPDR linkage interval in XLPDR-derived dermal fibroblasts (P1, P2, P3) was normalized against four normal fibroblast cell lines (F1, and three lines from unrelated males, U1, U2, U3). Only genes with detectable expression are displayed. (b) Immunoblotting determination of POLA1 protein expression in dermal fibroblasts from unaffected control lines and XLPDR-derived cells. (c) Quantitative RT-PCR (qRT-PCR) for POLA1 mRNA (left panel) in multiple independent samples from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines). POLA1 mRNA level is normalized to ACTB (×10−3). In addition, quantification of POLA1 protein levels shown in (b) are similarly presented (right panel). (d) Schematic representation of the mutation in intron 13 of the POLA1 gene. The location of primers used for RT-PCR are noted. Ex – exon. (e) RT-PCR using primers A and B (see d) and RNA from XLPDR-derived fibroblasts and a control line. The amplified products were separated by agarose gel electrophoresis and visualized with ethidium bromide. (f) qRT-PCR using transcript-specific primers for wild-type and misspliced forms of POLA1 mRNA from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines) was performed. (g) Exon trapping analysis in cells transfected with pSpliceExpress plasmids containing empty vector (EV), the wild type POLA1 intron 13 (A), or the intron bearing the XLPDR mutation (A→G). RT-PCR amplified products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining. *p<0.005, **p<0.001 (unpaired Student’s t-test). Data are pooled from 3 independent experiments (a), or representative from 2 (e,f,g) or 4 (b,e) independent experiments (mean and s.e.m.(a) or mean and individual sample values (c,f)).
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Figure 2: XLPDR is due to an intronic mutation that disrupts POLA1 expression(a) RNA-seq quantification of gene expression across the XLPDR linkage interval in XLPDR-derived dermal fibroblasts (P1, P2, P3) was normalized against four normal fibroblast cell lines (F1, and three lines from unrelated males, U1, U2, U3). Only genes with detectable expression are displayed. (b) Immunoblotting determination of POLA1 protein expression in dermal fibroblasts from unaffected control lines and XLPDR-derived cells. (c) Quantitative RT-PCR (qRT-PCR) for POLA1 mRNA (left panel) in multiple independent samples from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines). POLA1 mRNA level is normalized to ACTB (×10−3). In addition, quantification of POLA1 protein levels shown in (b) are similarly presented (right panel). (d) Schematic representation of the mutation in intron 13 of the POLA1 gene. The location of primers used for RT-PCR are noted. Ex – exon. (e) RT-PCR using primers A and B (see d) and RNA from XLPDR-derived fibroblasts and a control line. The amplified products were separated by agarose gel electrophoresis and visualized with ethidium bromide. (f) qRT-PCR using transcript-specific primers for wild-type and misspliced forms of POLA1 mRNA from dermal fibroblasts from unaffected individuals (8 samples, 4 cell lines) and XLPDR patients (10 samples, 2 cell lines) was performed. (g) Exon trapping analysis in cells transfected with pSpliceExpress plasmids containing empty vector (EV), the wild type POLA1 intron 13 (A), or the intron bearing the XLPDR mutation (A→G). RT-PCR amplified products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining. *p<0.005, **p<0.001 (unpaired Student’s t-test). Data are pooled from 3 independent experiments (a), or representative from 2 (e,f,g) or 4 (b,e) independent experiments (mean and s.e.m.(a) or mean and individual sample values (c,f)).
Mentions: Next, using dermal fibroblasts derived from three XLPDR probands and five control lines, we compared the expression levels of genes from the XLPDR linkage interval. Only POLA1 was significantly underexpressed in XLPDR cells (Fig. 2a). Furthermore, POLA1 protein expression in fibroblasts was markedly reduced as well (Fig. 2b). Quantitation using Li-COR indicated that POLA1 protein levels in XLPDR fibroblasts were reduced to about 35% of that seen in wild-type cells, and this was closely matched by the reduction observed by qRT-PCR at the mRNA level (Fig. 2c). An analogous effect on mRNA and protein expression was noted in patient-derived lymphoblastoid cell lines (LCLs, Supplementary Fig. 1d,e,f). Despite having reduced POLA1 levels, mutant fibroblasts did not display altered proliferative capacity or changes in cell cycle distribution (Supplementary Fig. 2a,b). Similarly, mitogen-induced lymphocyte proliferation was unaffected in two probands from the Waco, Texas family (Supplementary Fig. 2c). Furthermore, POLA1 silencing by siRNA to a degree comparable to that seen in XLPDR did not lead to cell cycle arrest in HeLa cells with an integrated cell-cycle reporter system (Supplementary Fig. 2d). Thus, the reduction of POLA1 expression in XLPDR did not appear to compromise cell replication, in agreement with the lack of a growth phenotype in most patients.

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.