High levels of rRNA synthesis by RNA polymerase I are important for cell growth and proliferation. In vitro studies have indicated that the formation of a stable complex between the HMG box factor [Upstream binding factor (UBF)] and SL1 at the rRNA gene promoter is necessary to direct multiple rounds of Pol I transcription initiation. The recruitment of SL1 to the promoter occurs through protein interactions with UBF and is regulated by phosphorylation of UBF. Here we show that the protein kinase CK2 co-immunoprecipitates with the Pol I complex and is associated with the rRNA gene promoter. Inhibition of CK2 kinase activity reduces Pol I transcription in cultured cells and in vitro. Significantly, CK2 regulates the interaction between UBF and SL1 by counteracting the inhibitory effect of HMG boxes five and six through the phosphorylation of specific serines located at the C-terminus of UBF. Transcription reactions with immobilized templates indicate that phosphorylation of CK2 phosphoacceptor sites in the C-terminal domain of UBF is important for promoting multiple rounds of Pol I transcription. These data demonstrate that CK2 is recruited to the rRNA gene promoter and directly regulates Pol I transcription re-initiation by stabilizing the association between UBF and SL1.

Control of ribosome biogenesis is a potential mechanism for the regulation of cell size during growth, and a key step in regulating ribosome production is ribosomal RNA synthesis by RNA polymerase I (Pol I). In humans, Pol I transcription requires the upstream binding factor UBF and the selectivity factor SL1 to assemble coordinately on the promoter. UBF is an HMG box-containing factor that binds to the rDNA promoter and activates Pol I transcription through its acidic carboxy-terminal tail. Using UBF (284-670) as bait in a yeast two-hybrid screen, we have identified an interaction between UBF and TAF1, a factor involved in the transcription of cell cycle and growth regulatory genes. Coimmunoprecipitation and protein-protein interaction assays confirmed that TAF1 binds to UBF. Confocal microscopy showed that TAF1 colocalizes with UBF in Hela cells, and cell fractionation experiments provided further evidence that a portion of TAF1 is localized in the nucleolus, the organelle devoted to ribosomal DNA transcription. Cotransfection and in vitro transcription assays showed that TAF1 stimulates Pol I transcription in a dosage-dependent manner. Thus, TAF1 may be involved in the coordinate expression of Pol I- and Pol II-transcribed genes required for protein biosynthesis and cell cycle progression.

Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to ϳ34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

Lin CH, Platt MD, Ficarro SB, Hoofnagle MH, Shabanowitz J, Comai L, Hunt DF, Owens GK. Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor. Am J Physiol Cell Physiol 292: C1617-C1624, 2007. First published December 20, 2006; doi:10.1152/ajpcell.00176.2006.-rRNA transcription is a fundamental requirement for all cellular growth processes and is activated by the phosphorylation of the upstream binding factor (UBF) in response to growth stimulation. Even though it is well known that phosphorylation of UBF is required for its activation and is a key step in activation of rRNA transcription, as yet, there has been no direct mapping of the UBF phosphorylation sites. The results of the present studies employed sophisticated nanoflow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-ESI-MS/MS) coupled with immobilized metal affinity chromatography (IMAC) and computer database searching algorithms to identify 10 phosphorylation sites on UBF at serines 273, 336, 364, 389, 412, 433, 484, 546, 584, and 638. We then carried out functional analysis of two of these sites, serines 389 and 584. Serine-alanine substitution mutations of 389 (S389A) abrogated rRNA transcription in vitro and in vivo, whereas mutation of serine 584 (S584A) reduced transcription in vivo but not in vitro. In contrast, serine-glutamate mutation of 389 (S389E) restored transcriptional activity. Moreover, S389A abolished UBF-SL1 interaction in vitro, while S389E partially restored UBF-SL1 interaction. Taken together, the results of these studies suggest that growth factor stimulation induces an increase in rRNA transcriptional activity via phosphorylation of UBF at serine 389 in part by facilitating a rate-limiting step in the recruitment of RNA polymerase I: i.e., recruitment of SL1. Moreover, studies provide critical new data regarding multiple additional UBF phosphorylation sites that will require further characterization by the field.immobilized metal affinity chromatography; mass spectrometry; UBF; TBP; RNA polymerase I ACTIVATION of rRNA transcription is required for sustained growth of all cells. In vitro, eukaryotic rRNA transcription can be reconstituted by the addition of three factors: RNA polymerase I (Pol I), selectivity factor 1 (SL1), consisting of the TATA binding protein (TBP), and three Pol I-specific TBPassociated factors (48, 63, and 110) and the upstream binding factor (UBF). It has been well recognized for nearly a decade that UBF is a necessary component for the activation of efficient rRNA transcription, and that phosphorylation of UBF dramatically enhances rRNA transcription in vitro. Furthermore, UBF phosphorylation is upregulated in states of cellular growth, consistent with a model whereby phosphorylation of UBF is a key mechanism linking cellular growth to activation of rRNA transcription. However, relatively little is known regarding the mechanisms by which UBF phosphorylation regulates transcriptional activity. Previous studies in our laboratory (17...