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.RNA is a highly structured macromolecule whose secondary and tertiary conformations facilitate an array of specific interactions with proteins. The DEAD/DEAH-box family of RNA helicases (here referred to as DDX/DHX) (3) is one such classification of RNA binding proteins that are capable of modifying the higher-ordered structures of RNA through the hydrolysis of ATP/nucleoside triphosphate (NTP) (41). DDX/ DHX proteins often form large multiprotein complexes that participate in fundamental biological activities such as RNA transcription, RNA editing, pre-mRNA splicing, ribosome biogenesis, and RNA decay (3).DDX/DHX helicases are named and characterized by the conserved DEAD/DEAH motif common among all family members. Through site-directed mutagenesis analysis, DEAD/ DEAH along with seven conserved peptide motifs have been found to participate in ATP/NTP binding, hydrolysis, and substrate binding (28). Despite the conservation of these peptide motifs, the remaining sequences within each RNA helicase family member vary widely. Specifically, differences exist between the two categories of DDX and DHX proteins. DDX proteins contain a unique Q motif at their N termini that distinguishes them from DHX proteins. It was proposed previously that the Q motif might sense the state of ATP in vivo (40), given that DHX-box proteins are promiscuous in their ability to utilize NTP (16).Ribosome biogenesis is a complex multistep process, the majority of which occurs in the nucleolus of the cell (24, 43). The transcription of ribosomal DNA (rDNA) is the initial and rate-limiting step in ribosome biogenesis, and as such, it is influenced by multiple levels of regulation (25). One of the key regulators of rDNA transcription is the upstream binding factor (UBF), a transcriptional transactivator that binds to the upstream core element of rDNA and subsequently bends rDNA (37). This change in the rDNA structure favors the binding of SL.1 as wel...

The role of exportin 6 in cytoskeletal-mediated cell death and cell adhesion in human non-small-cell lung carcinoma cells following doxorubicin treatment

“…Zhang et al showed the localization of FUrd in the nucleoli and connected this with active RNA polymerase I transcription [45]. On the other hand, Obrdlik and Percipalle observed the same localization of 5-FUrd as in the present study, and related this staining with polymerase II RNA.…”

DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle

“…Our study with lung cancer cells indicates that DHX33 deficiency causes cell cycle arrest or apoptosis independent of p53. We have previously found DHX33 to be a protein capable of associating with chromatin (3). In this study, we performed ChIP assays/EMSAs and found that DHX33 readily interacted with promoters of numerous important cell cycle-regulatory genes, whose transcriptional upregulation has been reported in human cancers.…”

“…Acute knockdown of DHX33 caused apoptosis in lung cancer cells but not in immortalized normal lung epithelial cells. We previously observed that in wild-type mouse embryonic fibroblasts (MEFs) and human primary fibroblasts, the majority of cells were arrested in G 1 /S-phase transition after DHX33 depletion (3,7) and that this was dependent on p53 function. Our study with lung cancer cells indicates that DHX33 deficiency causes cell cycle arrest or apoptosis independent of p53.…”

“…In previous studies, we identified DHX33 to be a significant player in ribosome RNA synthesis and mRNA translation (3,4). Other research groups have found DHX33 to be important in innate immunity as it can sense double-stranded RNA (dsRNA) from pathogens (5,6).…”

p19ARF and RasV12 Offer Opposing Regulation of DHX33 Translation To Dictate Tumor Cell Fate

“…We previously identified the nucleolar DHX33 DEAH-box RNA helicase as an important mediator of RNA Pol I transcription through its interaction with UBF at rDNA loci following serum stimulation (30). In the present study, we explored the mechanism underlying DHX33 regulation.…”