Our findings demonstrate that, in addition to controlling the transcription of rRNA and the translation of the mRNAs that encode ribosomal proteins, mTORC1 signaling also regulates the processing of pre-rRNAs in human cells. It thus exerts concerted control over multiple steps in ribosome biogenesis—rRNA transcription, pre-rRNA processing and the synthesis of ribosomal proteins. Such coordinated control over ribosome production makes clear physiological sense and is also consistent with the importance of ribosome production for cell growth and proliferation, cellular processes that are positively regulated by mTORC1 signaling.
We show that inhibition of mTORC1 by rapamycin interferes with at least distinct stages in pre-rRNA processes, the conversion of 30S pre-rRNA to the 21S intermediate and the processing of 32S rRNA, the precursor of the 28S and 5.8S rRNAs. AZD8055, a selective mTOR kinase inhibitor, appears to have additional effects pointing to roles for rapamycin-insensitive outputs from mTORC1 (25
) or perhaps a role for mTORC2 in pre-rRNA processing. It remains to be established how mTORC1 promotes pre-rRNA processing. Interestingly, rapamycin has also been shown to impair pre-rRNA processing in budding yeast (35
), indicating that control of this process by (m)TORC1 emerged early during eukaryotic evolution.
Since some Rps participate in the processing of rRNA (36
) and rapamycin inhibits the synthesis [reviewed in (4
)] of Rps, it was possible the changes in the levels of newly made r-proteins might be responsible for the effects of rapamycin on rRNA processing, as recently described in yeast (37
). Since there are about 80 different ribosomal proteins, it is not feasible to examine the potential involvement of each of them in the effects of mTORC1 inhibition on pre-rRNA processing all of them. Effects of rapamycin on the translation of the 5′-TOP mRNAs were quite modest. This contrasts with data from budding yeast where even very brief treatment with rapamycin (15
min) drastically decreased Rp synthesis (37
), perhaps reflecting the steep fall in Rp mRNA levels caused by rapamycin in this species [which probably arises because TORC1 promotes transcription of Rp genes in yeast, whereas it does not do so in mammals (35
)]. We also studied a possible role for RpS19, a protein that is mutated in Diamond-Blackfan anaemia, a condition which is associated with impaired ribosome biogenesis including the accumulation of early rRNA processing intermediates (36
). This indicates a key role for RpS19 in rRNA processing. However, overexpression of RpS19 did not overcome the defects in rRNA processing caused by rapamycin, indicating that the effects of mTOR inhibition cannot be attributed solely to possible changes in levels of this Rp (data not shown). Thus additional events are important in the control of rRNA processing downstream of mTORC1.
Our finding that mTORC1 is located in the nucleolus suggested a second potential mechanism by which rapamycin might affect rRNA processing; i.e. that affects the amount of mTORC1 associated with nucleoli. To study this, cells were pre-treated with rapamycin prior to fractionation. Rapamycin completely blocked the phosphorylation of RpS6 at Ser235/236 and Ser240/244 [the latter sites being specific targets for the S6 kinases (21
)], showing that it is effective in inhibiting mTORC1 signaling in the cellular compartments studied here (A). Rapamycin did not affect the distribution of GAPDH or lamin B (cytoplasmic and nuclear markers, respectively), but did cause a marked decrease in the amount of mTOR associated with nucleolar fraction, but not the nuclear fraction (A and B). This suggests that rapamycin causes the relocation of mTORC1 away from sites of active pre-rRNA processing, which may provide a mechanism by which it can regulate this process. The decrease in nucleolar mTOR levels may also play a role in the inhibitory effects of rapamycin on Pol I-mediated transcription (2
). It was clearly possible that the mTORC1 substrates S6K1 or (since we find it in the nucleus, E, albeit at low levels) S6K2 might play a role in rRNA processing. However, overexpressing S6K1 or S6K2 did not protect rRNA processing from inhibition by rapamycin, although it did eliminate its effect on RpS6 phosphorylation (data not shown). Clearly, further work is required to dissect the molecular events that underlie the control of pre-rRNA processing by mTORC1.
The observation that low levels of rapamycin (the minimum required to effectively inhibit mTORC1 signaling) impair uridine uptake suggests that mTORC1 signaling may promote the transport of nucleosides into mammalian cells; this would make good physiological sense as they are precursors for RNA and thus ribosome production, but further work is required to establish whether this effect genuinely reflects a role for mTORC1 signaling (as suggested by the fact that the inhibition caused by 5
nM rapamycin was almost maximal) or is an indirect or ‘off-target’ effect of this compound.