Identification of DHX33 as a Mediator of rRNA Synthesis and Cell Growth

Abstract

In this report, we employed a lentiviral RNA interference screen to discover nucleolar DEAD/DEAH-box helicases involved in RNA polymerase I (Pol I)-mediated transcriptional activity. Our screen identified DHX33 as an important modulator of 47S rRNA transcription. We show that DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci, where it interacts with the RNA Pol I transcription factor upstream binding factor (UBF). DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in levels of rRNA synthesis. Wild-type DHX33 overexpression, but not a DNA binding-defective mutant, enhanced 47S rRNA synthesis by promoting the association of RNA polymerase I with rDNA loci. In addition, an NTPase-defective DHX33 mutant (K94R) acted as a dominant negative mutant, inhibiting endogenous rRNA synthesis. Moreover, DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response, resulting in an attenuation of proliferation. Thus, we show the mechanistic importance of DHX33 in rRNA transcription and proliferation.

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ACKNOWLEDGMENTS

We thank the members of the Weber laboratory for their advice and technical assistance. The Genome Institute and Children's Discovery Institute at Washington University provided lentiviral RNAi library constructs.

J.T.F. was supported by NIH grant 5T32 GM007067. A.P.M. was supported by Department of Defense Breast Cancer Research Program award X81XWH-08-BCRP-PREDOC. This work was supported by NIH grant CA120436 and an Era of Hope scholar award in breast cancer research (BC007304) to J.D.W.