In last week’s blog, the role of FLCN in inhibiting rRNA synthesis was discussed. This week’s blog discusses a related study by Mekhail et al. (2006) that found rRNA synthesis can also be inhibited by nucleolar VHL.
The authors demonstrated that multiple murine (C2C12 and NIH3T3) and human (MCF7 and U‑87 MG) cell lines grown in non-buffered media under hypoxic conditions had higher ATP concentrations than those grown in standard media that prevents pH changes. Therefore, when hypoxic cells were able to acidify their environment – a process known as acidosis, where cells release lactic acid – they could maintain ATP levels better than cells that could not undergo acidosis. Cells synthesising lactic acid showed increased viability, as measured by fluorescein-diacetate and propridium iodide co-staining experiments. Hypoxic cells were treated with the glucose analogue 6‑deoxyglucose to inhibit ATP production by glycolysis; nonetheless ATP levels remained high, suggesting that these cells maintain their ATP levels by reducing energy consumption.
rRNA synthesis is believed to consume roughly 80% of a cell’s energy (Thomas, 2000), thus shutting down rRNA synthesis when resources are scarce is an efficient way to conserve energy and ensure cellular viability during hypoxia. Accordingly, fluorescence in situ hybridisation experiments using GFP-tagged UBF1 revealed that the nucleolar compartments containing rDNA, which encodes rRNA, were more condensed in cells undergoing acidosis, suggesting that hypoxia-induced acidosis reduces rRNA synthesis. This was confirmed by RT-PCR and pulse chase analysis measuring pre-rRNA synthesis.
The authors had previously observed that VHL re-localises to the nucleus during hypoxia-induced acidosis (Mekhail et al., 2004) and so wanted to investigate the involvement of VHL in rRNA silencing. Interestingly, VHL is required to mediate the reduction of rRNA synthesis during acidosis. Nucleoli of VHL-deficient cells (786-0) undergoing acidosis did not condense, with this phenotype being rescued by stably transfecting HA-tagged VHL into the cells. Confocal microscopy and photobleaching analysis of GFP-tagged VHL showed that it localises to the nucleolus and chromatin immunoprecipitation analysis showed VHL to be physically attached to rDNA during hypoxia-induced acidosis. Cells either lacking VHL or expressing a dominant negative form of the protein, which competes with wild type VHL for nucleolar localisation, could not maintain their ATP levels, leading to energy starvation and cell death.
As HIF1 is stabilised upon VHL’s translocation to the nucleus during acidosis (Mekhail et al., 2004), the authors investigated whether HIF is required for rDNA silencing by VHL. While the rate of acidosis was reduced, nucleolar condensation was still observed once the pH in the cell was sufficiently lowered, indicating that although HIF enhances rDNA silencing by VHL, it is not necessary.
There are striking similarities between this study and that of Gaur et al., (2012) discussed in last week’s blog. Firstly, both VHL and FLCN localise to the nucleolus and regulate rRNA synthesis. Thus it would be interesting to see whether rRNA synthesis inhibition by FLCN also occurs during hypoxia-induced acidosis and whether this involves VHL. This role in limiting cellular energy usage is also consistent with the known energy sensing role of FLCN (Baba et al., 2006). Secondly, Rpt4, which interacts with FLCN to reduce rRNA synthesis, is a proteasomal protein and VHL is known to target HIF for degradation in the proteasome, indicating that there may be functional overlap between these pathways; the proteolysis pathway’s role in clear cell RCC has also been discussed in a previous blog post.
- Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
- Gaur K, Li J, Wang D, Dutta P, Yan SJ, Tsurumi A, Land H, Wu G, & Li WX (2012). The Birt-Hogg-Dube tumor suppressor Folliculin negatively regulates ribosomal RNA synthesis. Human molecular genetics PMID: 23077212
- Mekhail K, Gunaratnam L, Bonicalzi ME, & Lee S (2004). HIF activation by pH-dependent nucleolar sequestration of VHL. Nature cell biology, 6 (7), 642-7 PMID: 15181450
- Mekhail K, Rivero-Lopez L, Khacho M, & Lee S (2006). Restriction of rRNA synthesis by VHL maintains energy equilibrium under hypoxia. Cell cycle (Georgetown, Tex.), 5 (20), 2401-13 PMID: 17102617
- Thomas G (2000). An encore for ribosome biogenesis in the control of cell proliferation. Nature cell biology, 2 (5) PMID: 10806485