While ARF has been long appreciated for its abilities to positively regulate p53 levels in the cell (22
) and serve as a sensor of hyperproliferative signals (19
), the relatively low abundance of ARF in interphase cells implied that ARF functioned only as a cellular checkpoint against aberrant growth and proliferation signals. In this manner, only signals powerful enough to elicit increases in ARF protein expression would trigger an actual ARF response. This implies that basal ARF molecules, even at their low levels, must be antagonized or held in check for the cell to undergo proper cell cycle progression and cell growth regimens. Teleologically, this model seems justified, given the genomic organization of the Ink4a/Arf
locus where “leakiness” in p16INK4a or p19ARF transcription would have dire effects on the growth and survival of the cell (14
). It is widely held that this locus is repressed in mice and that only under conditions of extreme stress or oncogenic signaling is the locus transcribed to elicit a growth and proliferative arrest phenotype (51
). Here, we provide evidence that the physiologically low level of ARF has a regulatory role in nucleolar function and ribosome biogenesis. Indeed, as early as 4 days post-ARF knockdown by lentiviral shRNA infection, we observed changes in nucleolar morphology and function that are reminiscent of data from the Arf−/−
embryonic cells. This strongly supports the hypothesis that basal ARF consistently monitors and dynamically alters the nucleolar growth/suppression pathway on a day-to-day basis. We would now argue that basal ARF proteins must be maintained at some steady-state level to provide constant surveillance of nucleolar function. Given the great energy demands of the nucleolus (ribosome biogenesis and protein synthesis account for nearly 50% of the cell's energy), dysfunctional nucleolar processes may need to be adjusted at a moment's notice (26
). In support of this contention, a recent report (33
) demonstrated that selective disruption of the nucleolus by either UV radiation or a number of “stress” responses induced cell cycle arrest and markedly enhanced p53 stability. While we did not observe any gross disruption of nucleoli in cells either lacking or overexpressing ARF, we did observe numerous qualitative changes in the size and number of nucleoli in cells lacking Arf
. This would suggest that basal ARF might play a vital role in determining the protein composition of nucleoli, acting to prevent the release of specific ribosomal proteins from the nucleolus or to prohibit the entrance of unwanted (potentially oncogenic) nuclear proteins into the nucleolus.
In the past few years, numerous p53-independent functions have been ascribed to ARF (35
). We found that nearly half of the basal ARF in the cell is in a complex with NPM, a protein previously shown to interact with human and mouse ARF proteins (4
). While much of the work concerning the ARF-NPM interaction has focused on the ability of each protein to antagonize the function of the other (4
), our findings suggest that the baseline interaction functions to maintain a controlled level of ribosome biogenesis. We propose a model where basal ARF antagonizes a small pool of NPM, either directly or enzymatically (8
), and thereby constantly limits ribosome output from the nucleolus. Importantly, levels of NPM did not change in the absence of ARF, but rather NPM activity was greatly increased as measured by its ability to promote ribosome nuclear export. Consistent with this model, the knockdown of basal NPM proteins resulted in dramatic reductions in protein production independent of cell proliferation, again underscoring the need for a consistent level of “ARF-free” NPM to promote ribosome synthesis.
While the mechanism and nature of such inhibition are still unclear, our data are consistent with a “thermostat” function for ARF, in that small changes in the abundance of ARF cause its binding partners to either dampen or enhance ribosome synthesis and export and, ultimately, lead to global changes in protein synthesis. It is apparent from our data that basal ARF can act in three distinct steps: (i) rDNA transcription, (ii) rRNA processing, and (iii) rRNA nuclear export.
While NPM has been ascribed roles in both rRNA processing and nuclear export (36
), we are uncertain of its ability to regulate rDNA transcription. In fact, NPM and ARF are both found in the granular region of the nucleolus, relatively far removed from the sites of nucleolar rDNA transcription (8
). However, we did observe significantly enhanced transcription of 47S rRNA in the absence of Arf
, implying that ARF proteins might regulate this process either directly or indirectly. This is not unprecedented, given recent findings that human ARF interacts with topoisomerase I to inhibit rDNA transcription (3
). Additionally, nearly half of the basal ARF protein is not bound to NPM, and we therefore cannot rule out the possibility that this pool of ARF is bound to proteins involved in rDNA transcription.
We suggest that ARF is expressed at a low level in interphase cells to ensure that proper growth control is achieved. This would serve to keep the cell in metabolic check, preventing the cell from wasting energy on unnecessary protein synthesis. Disruption of this exquisite basal ARF control would then have a twofold effect: (i) cells would produce far too many ribosomes, resulting in tremendous gains in protein synthesis, and (ii) the resultant cells would be highly susceptible to oncogenic signals. This setting would seem to provide a selective advantage to premalignant cells by ramping up their growth and, in the presence of appropriate signals, their proliferation. In support of this hypothesis, a recent study on the methylation of key loci involved in colorectal carcinogenesis demonstrated that 32% of the adenomas (premalignant lesions) isolated from patients with sporadic colorectal cancer demonstrated abnormalities at the Arf
). Our findings represent a novel and important role for basal ARF in maintaining protein synthetic homeostasis in nonmalignant cells. While NPM is certainly required for much of the ribosome biogenesis gains observed for Arf
-deficient cells, other interesting nucleolar targets of basal ARF must certainly exist. Precise details of how they may be affected remain elusive. Understanding the nucleolar integration of disparate requirements for proliferation, growth, and ribosome biogenesis will deepen our knowledge of how proteins like ARF adapted from regulators of cellular homeostasis to bona fide tumor suppressors.