A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10.
Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-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 mini-gene 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.
Yu
Qingming
Q
Department of Medicine and Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Guo
Jun
J
Zhou
Jianhua
J
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D005091:Exons / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D007438:Introns / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D024801:Tauopathies / Q000235:genetics; D014162:Transfection; D016875:tau Proteins / Q000235:genetics*
D012322:RNA Precursors; D016601:RNA-Binding Proteins; C491388:SRSF3 protein, human; D016875:tau Proteins; D000068103:Serine-Arginine Splicing Factors; D008156:Luciferases