Earlier this year, it was reported that FLCN, along with its interacting partners FNIP1 and FNIP2, regulates stem cell exit from pluripotency. In stem cells, the pluripotency transcription factor TFE3 was found in the nucleus and able to activate target genes. Upon differentiation signals, FLCN, FNIP1 and FNIP2 together exclude TFE3 from the nucleus, thus working as a molecular switch, allowing cells to exit the pluripotent state and become primed for differentiation (Betschinger et al., 2013). A recent study by Venables et al. may shed light on how this molecular switch may work.
Building on previous work, Venables et al. used a high throughput RT-PCR screen to find genes that are alternatively spliced during fibroblast reprogramming and differentiation. The genes MBNL1 and RBFOX2 were found to control nearly all the alternative splicing events observed during late mesoderm differentiation, including the alternative splicing of FNIP1, which is controlled by MBNL1.
In primary fibroblasts, a shorter isoform of FNIP1 – lacking the final 84 bp of exon 6, corresponding to amino acids 208-235 – predominated. Reprogramming of these fibroblasts to induced pluripotent stem cells (iPSCs) caused the longer, canonical, isoform of FNIP1 to predominate. This splicing pattern was fully reversed upon in vitro differentiation of iPSCs into fibroblasts.
Little is known about the structure of the FNIP1 protein, although it has recently reported to carry a non-canonical, interrupted DENN domain, and that the whole protein seems to be required to efficiently bind FLCN (Baba et al., 2006). Thus, how the function of the shorter FNIP1 isoform is affected by the loss of amino acids 208-235 is unknown. However, it has been previously reported that FNIP1-null mice have defective B-cells, which could provide further evidence that FNIP1 is required for late differentiation of mesodermal cell lineages.
Given that the full, canonical FNIP1 isoform is found in pluripotent cells and the shorter isoform is seen in differentiated cells, it could be that FNIP1 and FLCN are required to inhibit pluripotency and to promote differentiation, as reported in the Betschinger et al. study. Then once differentiation is complete, the shorter isoform of FNIP1 can no longer bind FLCN, thus inhibiting their function in terminally differentiated cells. Alternatively, it could be that the sequence spliced out of the shorter FNIP1 isoform is an inhibitory sequence, thus meaning that FNIP1 and FLCN function is inhibited in differentiating cells, but that they are active in differentiated cells. FLCN has been reported to inhibit the cell cycle (Laviolette et al., 2013, Lu et al., 2011, Nahorski et al., 2012), and thus may slow cell division in fully differentiated cells, as opposed to differentiating cells which divide more rapidly.
Ontological analysis of the 15 genes identified in this screen showed a preponderance of genes with important roles in membrane dynamics, polarity, cell adhesion and migration. Interestingly, FLCN has been implicated in all of these cellular processes, indicating that alternative splicing of FNIP1 may modulate FLCN’s function in these processes.
Finally, sequence analysis across species showed that 12 of the 15 alternatively spliced exons identified in this study were highly conserved throughout evolution. Furthermore, at least 8 of these 12 exons, including the FNIP1 exon, arose during the emergence of jawed vertebrates, indicating that alternative splicing of these genes may play an important role in the evolution and physiology of jawed vertebrates.
It is currently unclear how common alternative splicing of FNIP1 is and whether it plays any role in BHD pathogenesis. However, aberrant splicing is known to cause a number of human diseases, for example, mutations in MBNL1, which controls the alternative splicing of FNIP1, cause myotonic dystrophy DM1. Thus it is important to elucidate how alternative splicing of FNIP1 effects its binding to FLCN, and also how this shorter isoform may effect the function of FLCN itself.
- 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
- Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, & Smith A (2013). Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell, 153 (2), 335-47 PMID: 23582324
- Laviolette LA, Wilson J, Koller J, Neil C, Hulick P, Rejtar T, Karger B, Teh BT, &
Iliopoulos O (2013). Human folliculin delays cell cycle progression through late S and G2/M-phases: effect of phosphorylation and tumor associated mutations. PloS one, 8 (7) PMID: 23874397
- Lu X, Wei W, Fenton J, Nahorski MS, Rabai E, Reiman A, Seabra L, Nagy Z, Latif F, & Maher ER (2011). Therapeutic targeting the loss of the birt-hogg-dube suppressor gene. Molecular cancer therapeutics, 10 (1), 80-9 PMID: 21220493
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics, 21 (24), 5268-79 PMID: 22965878
- Venables JP, Lapasset L, Gadea G, Fort P, Klinck R, Irimia M, Vignal E, Thibault P, Prinos P, Chabot B, Abou Elela S, Roux P, Lemaitre JM, & Tazi J (2013). MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation. Nature communications, 4 PMID: 24048253
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.