TDP-43 differentially splices FNIP1

Folliculin interaction protein 1 (FNIP1), through interactions with FLCN, plays a role in a range of cellular processes (Baba et al., 2006). Alternative splicing of FNIP1, under the control of MBNL1, was previously reported in late mesenchymal differentiation (Venables et al., 2013). New research from De Conti et al., (2015) has identified FNIP1 as also being differentially spliced by TDP-43 – a protein associated with neurodegeneration in ALS and fronto-temporal dementia (Neumann et al., 2006).

TDP-43, a hnRNP component, helps regulates both DNA expression and mRNA processing, transport and translation for a wide range of genes (Buratti & Baralle, 2012). The loss of TDP-43 has previously been reported to alter expression and splicing of hundreds of genes (Polymenidou et al., 2011). However, as hnRNPs predominantly regulate splicing in a cooperative manner, it is unlikely that all of these genes are directly affected by TDP-43 levels. In fact very few genes have been shown to be directly affected by TDP-43; these include POLDIP3, CFTR, BIM, BCL2 and TARDBP itself.

De Conti et al. used HEK293 cells in a high throughput screen to identify changes in mRNA splicing directly related to TDP-43 depletion. Endogenous TDP-43 was knocked down using siRNA before attempted rescue with a siRNA-resistance wild type TDP-43 or F4L substitution mutant TDP-43 which cannot bind RNA. Using junction arrays it was possible to identify altered mRNA isoform production; genes that showed altered isoform production in the TDP-43 knockdown cells that could be rescued by TDP-43WT but not TDP-43F4L were deemed to be directly affected. In total 145 genes were identified including a large number involved in RNA binding and splicing, and several associated with apoptosis or cell cycle control.

Only the genes with at least a two-fold change in isoform profile, detectable by RT-PCR, were further characterised – of these the novel genes were STAG2, MADD, FNIP1 and BRD8. Altered splicing due to TDP-43 depletion results in the inclusion of STAG2 exon 30b and BRD8 exon 20, and the exclusion of FNIP1 exon 7 amino acids. However, exclusion of MADD exon 31 forms a premature stop codon leading to truncation and nonsense mediated decay. Using a second loss-of-function TDP-43 mutant (12XQ/N), which better mimics ALS pathology by inducing cytoplasmic aggregation of TDP-43 leading to nuclear depletion, resulted in similar alterations in isoform production. These alternations were also seen in neuroblastoma lines potentiality indicating a relevance in neuronal cells and new starting points for novel therapeutic strategies.

The results reported by De Conti et al. also support roles for TDP-43 in other cellular processes and potentially pathologies including cancer. MADD and STAG2 have roles in regulating apoptosis and cellular division respectfully, and have both been implicated in cancers (Kurada et al., 2009, Postal-Vinay et al., 2012).  FNIP1, through its interactions with FLCN and downstream signalling pathways, plays a role in energy metabolism, cell proliferation and apoptosis – processes perturbed in BHD tumours and other pathologies. The loss of FNIP exon 7 would not be expected to directly disrupt binding to FLCN or AMPK, as this is dependent on the C-terminal (Baba et al., 2006), but there are several phosphorylation sites within this domain that would be lost, potentially altering protein stability and interactions. Further research is required to determine what role, if any, TDP-43-depletion and the associated changes are playing in varied pathologies.

It is unknown whether alternative splicing of FNIP1 occurs in all tissues or whether it has any role in BHD pathology. Potentially different FNIP1 isoforms could unable to stably interact with FLCN, therefore perturbing FLCN-regulated cellular pathways leading to tumourigenesis. However, to date there have been no reports regarding TDP-43 or MBNL1 expression in BHD cells. As high throughput screens and genetic sequencing become more common in research it is quite likely that more interactions between BHD-related and novel proteins will be determined. Such discoveries could increase understanding of BHD pathogenesis and help in the development of new treatments.

  • 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. Proc Natl Acad Sci U S A. Oct 17;103(42):15552-7. PMID: 17028174.
  • Buratti E, Baralle FE. TDP-43: gumming up neurons through protein-protein and protein-RNA interactions. Trends Biochem Sci. 2012 Jun;37(6):237-47. PMID: 22534659.
  • De Conti L, Akinyi MV, Mendoza-Maldonado R, Romano M, Baralle M, Buratti E (2015). TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways. Nucleic Acids Res. Aug 10. pii:gkv814. [Epub ahead of print] PMID: 26261209.
  • Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. Oct 6;314 (5796):130-3. PMID: 17023659.
  • Kurada BR, Li LC, Mulherkar N, Subramanian M, Prasad KV, Prabhakar BS (2009). MADD, a splice variant of IG20, is indispensable for MAPK activation and protection against apoptosis upon tumor necrosis factor-alpha treatment. J Biol Chem. May 15;284(20):13533-41.PMID: 19289468.
  • Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW (2011). Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. Apr;14(4):459-68. PMID: 21358643.
  • Postel-Vinay S, Véron AS, Tirode F, Pierron G, Reynaud S, Kovar H, Oberlin O, Lapouble E, Ballet S, Lucchesi C, Kontny U, González-Neira A, Picci P, Alonso J, Patino-Garcia A, de Paillerets BB, Laud K, Dina C, Froguel P, Clavel-Chapelon F, Doz F, Michon J, Chanock SJ, Thomas G, Cox DG, Delattre O (2012). Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma. Nat Genet. Feb 12;44(3):323-7. PMID: 22327514.
  • 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. Nat Commun. 4:2480. PMID: 24048253.
Share This