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

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 synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I–Rrn3p levels.

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

“…The latter scenario is plausible since overexpression of wild-type Rrn3 was sufficient to increase rRNA synthesis under certain conditions in yeast and Drosophila [48,52]. In our study, however overexpression of Rrn3 enhanced Pol I transcription initiation, but it was unable to promote full-length rRNA production most likely because elongation or processes coupled to elongation such as processing become…”

“…On the other hand, in yeast cells TOR inactivation resulted in proteasomedependent degradation of Rrn3p and a strong reduction in Pol I transcription initiation [48]. Indeed, we found that serum withdrawal led to significant reduction in Rrn3 protein abundance within 30min (Fig 6A).…”

Reduction in Ribosomal Protein Synthesis Is Sufficient To Explain Major Effects on Ribosome Production after Short-Term TOR Inactivation in Saccharomyces cerevisiae

“…Since then, various studies have explored the molecular mechanism underlying this observation. It could be shown that the amount of initiation-competent complex of RNA Pol I with the essential transcription factor Rrn3 is reduced in rapamycin-treated cells (11,56). This observation correlated well with a reduced RNA Pol I association with the rRNA gene locus under these conditions (11,56).…”

“…Blot analysis and quantitation were performed as described elsewhere (52,56). For Northern blot analysis membranes were hybridized with a 32 P-labeled 25S oligonucleotide probe (probe 212, 5Ј-CTC CGC TTA TTG ATA TGC-3Ј).…”

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

“…When detached from DNA, free monomeric Pol I cannot directly be recruited to rDNA promoters but requires prior binding to the activating factor Rrn3 [12,13]. This event allows direct downregulation of rRNA production by Rrn3 degradation [24,30]. In addition, rDNA transcription is also down-tuned by Pol I dimerization [24].…”