DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner

Abstract

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.

Figure 1

RNA-dependent co-immunoprecipitation of NDH II with Ku antigen from HeLa nuclear extracts. (A) Co-immunoprecipitation of NDH II with Ku86 by a rabbit polyclonal antibody against NDH II. (B) Co-immunoprecipitation of Ku 86/70 with NDH II and DNA-PKcs by a mouse monoclonal antibody against Ku86. To digest RNA the thrice washed immunoprecipitates were incubated with RNase A (0.1 mg/ml) for 15 min at room temperature. After this treatment the immunoprecipitates were washed one more time and processed for western blotting. The immunoglobulin heavy chain (IgH) and light chain (IgL) are indicated.

Figure 2

RNA binding of Ku antigen in nuclear extract. (A) Gel filtration chromatography of HeLa nuclear extracts (2 ml) on Sepharose 6B. Elution of Ku antigen and NDH II after digestion of nuclear extracts by MNase (150 U/ml) plus RNase A (0.1 mg/ml) or by MNase (150 U) only for 15 min at room temperature was shown by western blotting (Aa). The multiple elution peaks of Ku86 from MNase plus RNase A treated nuclear extracts are indicated. The Sepharose 6B column was calibrated with the indicated molecular weight standards. Nucleic acids from the nuclease-treated extracts are shown by agarose gel electrophoresis (Ab). (B) RNAs were immunoprecipitated from 0.35 M NaCl nuclear extracts by the antibody against Ku86 and labeled with T4 RNA ligase and [5′-³²P]pCp after protein extraction with phenol–chloroform and RNA precipitation with ethanol. After labeling, the solutions were divided for digestion with RNase A as indicated. DNase I (0.1 mg/ml) was always present to remove possible contaminating DNA fragments. The labeling mixtures were then separated by electrophoresis through a 7 M urea–8% polyacrylamide gel. The RNA length standard was obtained from Roche; the positions of small nuclear RNAs (U1, U2, U4, U5 and U6) are indicated.

Figure 3

Co-immunoprecipitation of hnRNP C, NDH II and Ku antigen. Immunoprecipitation was performed with a mouse monoclonal antibody against hnRNP C protein (4F4) from 0.35 M NaCl nuclear extracts, followed by western blotting to visualize hnRNP C, NDH II and Ku86. The conditions were as described in Figure 1. The immunoglobulin heavy chain (IgH) and light chain (IgL) are indicated.

Figure 4

Ku86 co-immunoprecipitated with hnRNP complexes from the nucleoplasm of HeLa cells. Nucleoplasmic extracts of HeLa cells were prepared after metabolic labeling with [³⁵S]methionine, followed by immunoprecipitations with a mouse antibody against (A) Ku86 or (B) hnRNP C. Proteins were separated by two-dimensional electrophoresis, followed by fluorography (Aa and B) or western blotting (Ab) with a mixture of antibodies against hnRNP K (P-20), hnRNP-F (N-15) that also cross-react with hnRNP H and hnRNP C (4F4) (Ab, upper panel) or only an antibody against hnRNP K (p20) (Ab, lower panel). Letters denote the different hnRNP proteins.

Figure 5

RNA-dependent phosphorylation of hnRNP proteins by DNA-PK. hnRNP proteins were obtained from HeLa nucleoplasmic extracts by immunoprecipitations with the antibody against hnRNP C. (A) DNA-PK catalyzed an RNA-dependent phosphorylation of hnRNP C and A1. Where indicated the samples were digested with RNase A (0.1 mg/ml) for 15 min at room temperature prior to the addition of DNA-PK. (B) The phosphorylation levels of hnRNP C and A1 were measured by scintillation counting of the excised protein bands and expressed as values relative to the background (no addition of DNA-PK). (C) Western blot of hnRNP C and A1 from hnRNP C immunoprecipitates eluted from protein-A agarose beads. RNase treatment was as described above.

Figure 6

RNA-dependent phosphorylation of NDH II by DNA-PK. (A) (Aa) DNA-PK phosphorylated NDH II in the presence of native RNA. Purified His₆-tagged NDH II (≈1 µg each) (23) was used as protein substrate for the DNA-PK assay. Poly(A)⁺-RNA was from mouse spleen (Clontech) and added to ≈0.1 mg/ml. DNA was from calf thymus and used at 0.05 mg/ml. RNase A treatment was as described in Figure 5. (Ab) Phosphorylation of NDH II in the presence of synthetic RNAs. DNA-PK was incubated with His₆-tagged NDH II (≈1 µg each) in the presence of poly(rA), poly(rC), poly(rG) and poly(rU). Phosphorylation levels relative to the background (i.e. in the absence of nucleic acids) are also presented. The signals were quantified with a PhosphorImager. (B) RNA binding assay of DNA-PK. (Ba) Filter binding of DNA-PK with the indicated ³²P-labeled ribopolymers poly(rA), poly(rC), poly(rG) and poly(rU). (Bb) Northwestern blot of the Ku heterodimer. Ku86/70 was obtained from HeLa nuclear extract by immunoprecipitation as described in Figure 1. After separation by SDS–PAGE the proteins were transferred to a Hybond-C nitrocellulose membrane (Amersham) and probed by the same RNAs as used for filter binding. Only the Ponceau S protein staining and the membrane probed with poly(rG) are shown. Ig, immunoglobulin.

Figure 7

Immunofluorescence of HeLa cells after transfection of siRNAs against NDH II and DNA-PK. siRNAs against the expression of (A) NDH II and (B) DNA-PKcs were transfected into HeLa cells that were attached to cover slips. Mock transfected controls were also carried out (Aa–c and Ba and b). Forty-eight hours after transfection immunofluorescence was performed with a rabbit antibody against NDH II (Ab, e and h; Bd), a mouse monoclonal antibody against hnRNP C (4F4) (Aa and d; Bh) or an antibody against the catalytic subunit of DNA-PK (DNA-PKcs) (Ag; Ba, c and g). (Bf) Phase contrast image of HeLa cells. DNA is shown by DAPI stainings in (Ac, f and i) and (Bb, e and i). Arrows indicate HeLa cells that exhibit siRNA silencing of NDH II- or DNA-PKcs expression.