Recent studies have implicated FLCN in numerous cellular pathways through the identification of novel interacting partners, such as Plakophilin-4 (which is discussed in a previous blog post). Another study has now demonstrated that the Drosophila homologue of FLCN (dFLCN) appears to regulate ribosomal RNA (rRNA) synthesis through its interaction with Regulatory particle triple-A ATPase 4 (Rpt4) (Gaur et al., 2012).
In this study, the authors identified Rpt4 as an interacting protein using full-length dFLCN as bait in a yeast two-hybrid system. Co-immunoprecipitation experiments using HEK293T cells confirmed this interaction, and immunostaining also showed that FLAG-tagged Rpt4 (Rpt4-FLAG) and HA-tagged dFLCN (dFLCN-HA) co-localised to the nucleus of Drosophila Schneider 2 (S2) cells. Further immunostaining analysis indicated that dFLCN-HA specifically co-localised with the nucleolar marker Fibrillarin, and that RNAi knockdown of dFLCN led to multiple-lobed nucleoli and an increase in nucleolar volume. These effects were rescued by dFLCN-HA over-expression.
The nucleolus and Rpt4 are known to be involved in rRNA synthesis (Ottosen et al., 2002; Fátyol & Grummt, 2008), so could dFLCN be involved in this process too? In fly larvae, RNAi-mediated knockdown of dFLCN led to a 3-5-fold increase in pre-rRNA levels, and over-expression of dFLCN-HA marginally decreased pre-rRNA levels, as measured by qRT-PCR and in situ hybridisation. Given this, could dFLCN affect rRNA synthesis through Rpt4? Chromatin immunoprecipitation experiments using S2 cells showed that dFLCN-HA over-expression reduced Rpt4 binding to rDNA in comparison to controls.
To understand the contribution of this interaction to tumourigenesis, Gaur et al. also showed that dFLCN RNAi-knockdown enhanced Ras-induced cell proliferation, eye deformation and lethality in a fly with constitutively active Ras1 in the eye. dFLCN-HA over-expression was able to mostly rescue the eye and cell proliferation phenotypes, but had no significant effect on lethality. In contrast, reduced expression of Rpt4 increased the viability of these flies, while over-expression decreased their viability. These results confirm that FLCN plays a role in the Ras/ERK pathway (as illustrated in our signalling diagram) and that rRNA synthesis may also be involved in tumour formation.
Finally, immunostaining experiments using polyclonal antibodies (raised against a synthetic polypeptide which relates to the C-terminal region of full-length human FLCN) showed that the nucleolar localisation of FLCN was conserved in normal human skin and kidney tissues, as well as HeLa cells. Furthermore, it could be seen that the reintroduction of FLCN into FLCN-null UOK257 cells led to a 2-fold decrease in pre-rRNA levels, as well as fewer ribosomal subunits. shRNA knockdown of Rpt4 in UOK257 cells had no effect on cell growth and small effects on colony growth in soft agar in vitro, however, there was decreased tumour growth in xenograft experiments in mice, when compared to controls. Consequently, could Rpt4 inhibition present a novel avenue for tumour suppression in BHD syndrome? In addition, this study shows interesting parallels to a study by Mekhail et al. (2006), in which nucleolar VHL regulates rRNA synthesis during hypoxia (which will be discussed in more detail in the near future).
- Fátyol K, & Grummt I (2008). Proteasomal ATPases are associated with rDNA: the ubiquitin proteasome system plays a direct role in RNA polymerase I transcription. Biochimica et biophysica acta, 1779 (12), 850-9 PMID: 18804559
- 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, 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
- Ottosen S, Herrera FJ, & Triezenberg SJ (2002). Transcription. Proteasome parts at gene promoters. Science (New York, N.Y.), 296 (5567), 479-81 PMID: 11964465