Growth-dependent regulation of ribosome biogenesis and the role of Rrn3p in RNA polymerase I transcription

Eukaryotic cell growth is tightly linked to the synthesis of new ribosomes, the molecular machineries responsible for protein production. The transcription of a ribosomal precursor RNA (pre-rRNA) by RNA polymerase I (Pol I) constitutes an initial and central step in the complex process of ribosome biogenesis and is therefore one of the main targets for regulation. The initiation of each round of transcription is dependent on the formation of a complex between Pol I and the essential transcription factor Rrn3p. Subsequent processing of this precursor transcript yields three of the four mature ribosomal RNAs (rRNAs) forming a scaffold to which ribosomal proteins (r-proteins/RPs) assemble in the course of ribosome maturation. Since ribosome biogenesis is one of the most energy-consuming cellular processes, eukaryotic cells cease the production of ribosomes very rapidly upon unfavorable growth conditions like nutrient deprivation in order to ensure survival. The conserved target of rapamycin (TOR)-pathway plays an essential role in both sensing environmental changes and mediating adequate cellular responses. Inhibition of TOR complex 1 (TORC1) induces an immediate drop in the synthesis rate of ribosomes. It was previously suggested that TOR inactivation interferes with ribosome synthesis in many ways, but it was unclear whether and how these processes are coordinated. To distinguish between primary and secondary effects on ribosome biogenesis in the yeast Saccharomyces cerevisiae and to determine the target mediating the fast response to TOR inactivation, Pol I transcription and rRNA synthesis were investigated shortly after TOR inhibition by rapamycin. This drug mimics nutrient starvation of cells by specifically inactivating the kinase activity of TORC1. The following conclusions could be drawn: 1) A rather long-term response constitutes the decrease in the level of Rrn3p leading to less initiation-competent Pol I-Rrn3p complex formation and thus reduced Pol I transcription. Rrn3p is characterized by a short half-life which is due to its constitutive ubiquitin-dependent degradation. Consequently, the level of Rrn3p is quickly down-regulated when the neo-synthesis of the protein is inhibited. 2) The fast down-regulation of mature rRNA synthesis correlates with serious pre-rRNA processing defects and subsequent RNA degradation, but not with the inhibition of Pol I transcription, since the association of Pol I with the rRNA gene locus is yet unaltered and the Pol I molecules engaged in transcription are still mobile. 3) The quick down-regulation of r-protein synthesis is sufficient to explain the severe pre-rRNA processing defects. The strong decrease in general translation, presumably along with the specifically reduced transcription rate of ribosomal protein genes, seems to cause the drastic repression of r-protein production. Since the level of Rrn3p appears to play a crucial role in Pol I transcription in yeast, this issue was investigated in more detail. Interestingly, already scarce amounts of Rrn3p are sufficient to promote Pol I transcription and cell growth, whereas strong overexpression of this factor results in growth defects. Elevated levels of Rrn3p lead to enhanced Pol I-Rrn3p complex formation, however, the question whether the growth defect is caused by the concomitantly observed increase in pre-rRNA-levels remains to be elucidated. Finally, Pol5p, which was published to play an essential role in the synthesis of ribosomal RNA in yeast, co-purified with Rrn3p through several purification steps suggesting an interaction between the two proteins. However, further experiments provided only weak additional evidence for Pol5p as a genuine interaction partner of Rrn3p and failed to confirm the reported association of this protein with the rRNA gene locus. Therefore, further investigation is required to elucidate the role of Pol5p in ribosome biogenesis.