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.

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by cancer predisposition and idiopathic growth retardation. The BLM helicase, mutations of which lead to BS, localizes to the nucleolus of the cell, the site of RNA polymerase l-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. Disorders with defective ribosome biogenesis, such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome, display similar cancer predisposition and growth abnormalities to BS. The objective of this study was to investigate a potential mechanism underlying the proportional dwarfism of BS. We tested the hypothesis that the BLM helicase facilitates rRNA transcription as a component of an RNA polymerase I-associated complex, which when deficient has the potential to lead to proportional dwarfism. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription using H3-uridine pulse-chase assays and nuclear run-on assays in cells with an innate or experimentally induced BLM deficiency. In vitro helicase assays demonstrate that BLM unwinds GC-rich hybrid RNA/DNA nucleic acid substrates predicted to form in the nucleolus during rRNA transcription; the persistence of such structures inhibits progression of the RNA polymerase I transcription complex. Immunofluorescence studies using actinomycin D at a concentration that inhibits RNA polymerase I demonstrate the dependence of BLM nucleolar localization upon ongoing RNA polymerase l-mediated rRNA transcription. Finally, in vivo protein co-immunoprecipitation shows that BLM physically associates with RNA polymerase I in the nucleolus. These studies suggest that nucleolar BLM interacts with RNA polymerase I and facilitates rRNA transcription by modulating rDNA structure. Given the intricate relationship between rDNA metabolism and growth, our data may help to understand the etiology of the proportional dwarfism in Bloom's syndrome. Furthermore, this work provides greater understanding of the mechanisms regulating rRNA transcription and ribosome biogenesis, processes currently being exploited in cancer therapeutics. This abstract is also presented as Poster B34. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR3.