BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription

Abstract

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

Figure 1.

Nucleolar localization of BLM is dependent upon ongoing RNA polymerase I transcription. (A) The breast cancer cell line MCF7 was transiently transfected with pGFP–BLM and stained with αRPA194, a nucleolar protein, to demonstrate co-localization of GFP–BLM to nucleoli. MCF7 cells were treated with either actinomycin D (AMD), 4-nitroquinoline-1 oxide (4NQO), α-amanitin, hydroxyurea (HU), DMSO (negative control for AMD and 4NQO) or H₂O (negative control for α-amanitin and HU), followed by staining with αRPA194 and visualization of transiently expressed GFP–BLM and RPA194. MCF7 cells were transfected with pGFP control vector and similarly treated to demonstrate a lack of an effect on GFP. (B) The results of scoring transiently transfected MCF7 cells for GFP–BLM localization following the indicated treatments are shown. Averages ± standard deviation were calculated for a minimum of 60 cells per treatment. (C) MCF7 cells were transiently transfected with pGFP–BLM, treated with AMD, 4NQO, DMSO (negative control for AMD and 4NQO) or H₂O and stained with αPML to analyze the co-localization of GFP–BLM and PML when BLM dissociates from the nucleolus.

Figure 2.

BLM associates with the RNA polymerase I-specific subunit RPA194. (A) Co-immunoprecipitations were performed with nuclear extracts from 293T cells using either αBLM or αRPA194 antibodies for IP (51). Proteins were separated using 8% SDS–PAGE, blotted and analyzed with αBLM and αRPA194 antibodies; goat IgG is an isotype-matched negative control for αBLM, mouse IgG is an isotype-matched negative control for αRPA194. (B) Co-immunoprecipitations were performed as in (A) but αRPB1 (RNA polymerase II subunit) was used; mouse IgG is an isotype-matched negative control for anti-RNA polymerase II. In (B), we were unable to detect an interaction between BLM and RNA polymerase II, demonstrating the specificity of its interaction with RPA194 in (A).

Figure 3.

BLM deficiency slows RNA polymerase I-mediated 45S rRNA transcription rate. (A) 293T cells transfected with either an αBLM-directed siRNA or scrambled control siRNA were pulse-labeled with ³H-uridine for 30 min (P) and chased for 1 h (C) with cold uridine. Isolated RNAs were separated on a 1% MOPS-formaldehyde agarose gel, transferred to a nylon membrane and analyzed by autoradiography. 45S, 28S and 18S rRNA species are indicated next to autoradiograph (top panel). Ethidium bromide staining demonstrates equal loading of RNA (middle panel). Western blot demonstrates efficiency of BLM knockdown; lamin B serves as a protein loading control (bottom panel). (B) ³H-uridine pulse-chase analysis in BS fibroblasts (GM08505) transfected with either pGFP–BLM or pGFP; figure is labeled as in (A). (C) The pulse-chase analyses in 293T and GM08505 cells were analyzed using ImageQuant software to measure the 45S rRNA transcript abundance in the BLM-proficient cells (293T scrambled siRNA-transfected or GM08505 pGFP–BLM-transfected) compared with the BLM-deficient cells (293T αBLM-directed siRNA-transfected or GM08505 pGFP-transfected).

Figure 4.

BLM unwinds duplex substrates with a 3′ DNA overhang but not those with a 3′ RNA overhang. (A) The purity of BLM protein was determined using electrophoresis with 8% SDS–PAGE gels and staining with SYPRO Ruby Protein Gel Stain (Sigma). (B) Autoradiographs of representative gels illustrate unwinding activities. BLM (3.8 nm) was incubated with substrate for 0, 2.5, 5, 10, 20 or 30 min at 37°C as described in Materials and Methods. Products were resolved using 12% non-denaturing acrylamide gels. Unwinding is demonstrated by conversion of duplexed substrate to faster migrating single-stranded oligonucleotide. HD is heat-denatured substrate produced by heating to 95°C for 5 min. (C) Kinetics of BLM unwinding of RNA- and DNA-containing substrates. BLM unwinds DNA₂₀:DNA₃₃ and RNA₂₀:DNA₃₃ but does not appreciably unwind DNA₂₀:RNA₃₃ or RNA₂₀:RNA₃₃. Unwinding of each duplex substrate was calculated by comparing the amount of single-stranded substrate produced to the total amount of substrate in the reaction with correction for any un-annealed substrate in zero-time controls. Percent unwinding is graphed as a function of time.

Figure 5.

BLM binds to DNA₂₀:DNA₃₃ and RNA₂₀:DNA₃₃, and less strongly to DNA₂₀:RNA₃₃ and RNA₂₀:RNA₃₃ duplexes. (A) Purified BLM (0, 11, 15, 23, 30, 38, 56, 75, 110 nm) was incubated with ³²P-labeled substrates as described in Materials and methods. Reactions were separated using acrylamide gel electrophoresis and analyzed using ImageQuant software. Duplexes bound by BLM migrate more slowly (open circles) than unbound duplexes (asterisk). (B) Binding of each duplex was calculated by comparing the amount of bound complex to the total amount of duplex in the reaction. Percent binding is graphed as a function of BLM concentration. (C) Purified BLM (0, 13.3, 29, 40 and 55 nm) was incubated with ³²P-labeled single-stranded DNA₄₆ or RNA₄₆ as described in Materials and Methods. Reactions were separated using 1% agarose gels that were dried and analyzed. Substrate bound by BLM migrates more slowly (open circles) than unbound substrate (asterisk).

Figure 6.

Model for the role of BLM in rRNA transcription. During RNA polymerase I-mediated 45S rRNA transcription, RNA–DNA hybrids may form between the nascent rRNA transcript and the template rDNA (28). We propose that BLM unwinds the RNA–DNA hybrid so that transcription and replication proceed unaffected. In the absence of BLM, the RNA–DNA hybrid may remain unresolved resulting in retardation of further rRNA transcription, stalling of transcription and replication forks, and the induction of recombination within the rDNA (37).