A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

Abstract

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 , and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of minigene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing. Keywords: alternative splicing, frontotemporal dementia with parkinsonism associated with chromosome 17, progressive supranuclear palsy, serine-arginine (SR) proteins, tau, tauopathy. The human tau gene contains 16 exons and encodes a fulllength 441 amino-acid tau protein. However, three exons, namely 2, 3 and 10, undergo alternative splicing, giving rise to six different isoforms of tau proteins (van Slegtenhorst et al. 2000). The main function of tau proteins is to bind and stabilize microtubules (MTs) via their four carboxyl-terminal tandem repeat sequences of 31 or 32 amino acids Goedert et al. 1989;Andreadis et al. 1992). Alternative splicing of exon 10, which encodes the second repeat, produces 3-repeat tau (3R) or 4-repeat tau (4R) (Lee et al. 2001). 4R-tau isoforms have a greater MT-binding affinity and are more efficient at promoting MT polymerization than 3R-tau isoforms (Goedert and Jakes 1990;Goode and Feinstein 1994). In adult human brain, the ratio of 3R-tau to 4R-tau isoforms is approximately 50 : 50 (Goedert and Jakes 1990). Therefore, production of 4R or 3R proteins in a normal individual is expected to be functionally balanced. Disruption of this balance would possibly induce abnormality.Many neurodegenerativ...

Sex-dependent up-regulation of two splicing factors, Psf and Srp20, during hippocampal memory formation

“…Although the roles of PSF and SRP20 as splicing factors are well established (Patton et al 1993;Jumaa and Nielsen 1997;Shav-Tal and Zipori 2002;Galiana-Arnoux et al 2003;Sanford et al 2005), not much is known about the function of SRP20 and PSF in neurons. SRP20 regulates splicing of exon 10 of the gene encoding the microtubule-associated protein tau (Yu et al 2004), which might indicate a role for compartmentalized signaling during memory processes. The neuronal expression of Psf is established (Chanas-Sacre et al 1999), but the transcripts it splices in these cells remain to be determined.…”

“…In this study we demonstrated that the gene encoding the glycosyl phosphatidyl-inositol anchor attachment protein 1 (Gaa1) requires CaMKK␤ for up-regulation by spatial training in males (Mizuno et al 2007). Here, we show that two splicing factors, the splicing factor arginine/serine-rich 3 (Sfrs3/Srp20) (Jumaa and Nielsen 1997;Yu et al 2004) and polypyrimidine tract-binding (PTB) protein-associated splicing factor (Psf) (Chanas-Sacre et al 1999) are differentially expressed in the naïve male hippocampus of CaMKK␤-deficient mice. Since increasing evidence suggests that alternative splicing is an important, but not well understood mechanism of brain plasticity (Bottai et al 2002;Lee and Irizarry 2003;Nijholt et al 2004;O'Connor et al 2004;Rattiner et al 2004;Beffert et al 2005), we studied the expression of Srp20 and Psf in more detail.…”

Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

“…pCEP4/ SRp55-HA was a gift from Dr. Tarn of the Institute of Biomedical Sciences, Academia Sinica, Taiwan. pCI/SI9-LI10, containing a Tau mini-gene SI9-LI10, comprising Tau exons 9 -11 and part of intron 9 and the full length of intron 10, was described previously (11). Monoclonal antibody against Dyrk1A (8D9) was raised against a histidine-tagged protein containing the first 160 amino acid residues of rat Dyrk1A (18).…”

“…SRp55 (serine/arginine-rich protein 55) is a SR protein with an apparent molecular mass of 55 kDa and participates in constitutive and alternative pre-mRNA splicing (6). SRp55 was reported to regulate the alternative splicing of Bim (7), epidermal growth factor (8,9), HIV-1 (10), and Tau (11).…”

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

“…The splicing factors SRp20, SRp30c, SRp55, Tra2-b, and 9G8 have all been shown to affect exon 10 splicing. Each of these proteins contains an arginineserine repeat (RS) domain, characteristic of the SR family of splicing factors (Yu et al 2004;Wang et al 2005;Gao et al 2007). SR proteins can act as either alternative splicing enhancers or repressors (Long and Caceres 2009;Shepard and Hertel 2009).…”