Rrn3 Becomes Inactivated in the Process of Ribosomal DNA Transcription

Abstract

The human homologue of yeast Rrn3, a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription. Although the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined. It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I⅐Rrn3 complexes that can interact with the committed template. These and other reported differences would have implications with respect to the mechanism by which Rrn3 functions in transcription. For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically. Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically. We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed. Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions. For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction. We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I. Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription and that its ability to associate with RNA polymerase I is lost upon transcription.

Dephosphorylation of RNA Polymerase I by Fcp1p Is Required for Efficient rRNA Synthesis

“…Mammalian Rrn3p/TIFIA is a growthregulated factor in rDNA transcription (14) and was shown to associate with Pol I only in growing but not in quiescent or down-regulated cells (15)(16)(17)(18). Together, these findings suggest that reversible post-translational modification(s) of either Pol I or Rrn3p mediate their stable interaction.…”

“…Fcp1p might also modulate the phosphorylation pattern of the Rrn3p-Pol I complex after the formation of the preinitiation complex at the promoter has occurred. Because Rrn3p leaves Pol I during one round of transcription (13,18), Fcp1p activity might result in dissociation of the Rrn3p-Pol I complex, allowing the elongation-active Pol I to clear the promoter and continue RNA synthesis. However, our results indicate that Fcp1p neither affects formation nor dissociation of the free Rrn3p-Pol I complex, and it remains to be determined whether Fcp1p dissociates the Pol I-Rrn3p complex if the complex is bound to the promoter.…”

“…In contrast to the yeast system, the association of mammalian Rrn3p/TIFIA with Pol I is dependent on the phosphorylated form of Rrn3p (17,18). The activity of mammalian Rrn3p/ TIFIA is modulated by mitogen-activated protein kinases, thus connecting regulation of rDNA transcription to cellular growth and proliferation (20).…”

Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress

“…HeLa cells were transfected with FLAG-tagged RRN3 plasmid (30) with Effectene (Qiagen), and expressed proteins were purified from cell lysates in 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM PMSF using anti-FLAG agarose beads (Sigma). Beads were eluted with 0.5 mg/ml FLAG peptide (Sigma) and dialyzed with 20 mM HEPES (pH 8.0), 20% glycerol, 100 mM KCl, 5 mM MgCl 2 , 0.2 mM EDTA, 1 mM PMSF, and 0.5 mM dithiothreitol.…”

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

“…Rrn3 has been proposed as a major regulatory factor that links growth factors, nutrients and stress factor signalling to rDNA transcription rates through the formation of a transcriptionally competent Pol I complex [26][27][28][29][30][44][45][46][47]. More specifically, serine residues 44, 633 and 649 on Rrn3 were shown to be phosphorylated by mTOR, ERK and RSK signalling pathways, respectively [28,29].…”