Published online 25 April 2010 Nucleic Acids Research, 2010, Vol. 38, No. 16 5315–5326 doi:10.1093/nar/gkq264 TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production Anja Philippi, Robert Steinbauer, Alarich Reiter, Stephan Fath, Isabelle Leger-Silvestre, Philipp Milkereit*, Joachim Griesenbeck* and Herbert Tschochner* Institut fu¨ r Biochemie, Genetik und Mikrobiologie, Universita¨ t Regensburg, Universita¨ tsstr. 31, 93053 Regensburg, Germany Received February 25, 2010; Revised and Accepted March 30, 2010 ABSTRACT Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome- dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I–Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I–Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthe- sis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I–Rrn3p levels. INTRODUCTION A crucial step in the regulation of ribosome synthesis is the adjustment of ribosomal RNA (rRNA) gene transcription according to changes in the extracellular environment. Production of rRNAs depends on multiple signalling pathways responding to nutrient availability, stress stimuli or mitogen activation (1–3). One bona ﬁde target of the intracellular signal transduction pathways is the ribosomal gene transcription apparatus including RNA Pol I and associated transcription factors. Some of them like mammalian UBF, SL1 and Rrn3/TIF-IA were shown to be either aﬀected by mitotic silencing (4,5) or growth regulated by MAP-kinase (6–8) the mTOR- (target of rapamycin) (9–12), JNK- (13) and casein kinase II-pathways (14–16). These studies suggested that site speciﬁc phos- phorylation of single factors results in either enhanced or reduced formation of Pol I-complexes initiating tran- scription at the rDNA promoter. Furthermore, rapamycin-dependent inactivation of rRNA synthesis correlated with the dissociation of Rrn3p/TIF-IA from Pol I and with its translocation from the nucleolus to the cytoplasm (12). It was also proposed that UBF asso- ciation with the transcribed rDNA region might act as an obstacle for the elongating form of Pol I that can be overcome by growth factor-dependent phosphorylation (17). On the other hand, UBF was suggested to play a role in promoter escape (18). Thus, it appears that eukary- otic rDNA transcription can be regulated at many diﬀer- ent levels. *To whom correspondence should be addressed. Tel: +49 941 943 2846/2472; Fax: +49 941 943 2474; Email: herbert.tschochner@vkl.uni-regensburg.de Correspondence may also be addressed to Philipp Milkereit. Tel: +49 941 943 2846/2472; Fax: +49 941 943 2474; Email: philipp.milkereit@vkl.uni-regensburg.de Correspondence may also be addressed to Joachim Griesenbeck. Tel: +49 941 943 2846/2472; Fax: +49 941 943 2474; Email: joachim.griesenbeck@vkl.uni-regensburg.de Present addresses: Anja Philippi, KIST Europe Forschungsgesellschaft mbH, Campus E 7.1 66123 Saarbru¨ cken, Germany. Stephan Fath, GENEART AG, BioPark, Josef-Engert-Str. 11, 93053 Regensburg, Germany. Isabelle Leger-Silvestre, Laboratoire de Biologie Mole´ culaire Eucaryote, Universite´ Paul Sabatier, Toulouse III, France. The authors wish it to be known that, in their opinion, the ﬁrst three authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.