title: Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.
abstract: Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximately 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.
authors.affiliation: Medical Research Council Centre for Neurodegeneration, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, United Kingdom.
authors.forename: Teresa
authors.initials: T
authors.lastname: Rodriguez-Martin
authors.forename: Mariano A
authors.initials: MA
authors.lastname: Garcia-Blanco
authors.forename: S Gary
authors.initials: SG
authors.lastname: Mansfield
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Grover
authors.forename: Michael
authors.initials: M
authors.lastname: Hutton
authors.forename: Qingming
authors.initials: Q
authors.lastname: Yu
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhou
authors.forename: Brian H
authors.initials: BH
authors.lastname: Anderton
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Gallo
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism; D017381:Spliceosomes / Q000502:physiology*; D024801:Tauopathies / Q000209:etiology* / Q000235:genetics; D020040:Trans-Splicing; D016875:tau Proteins / Q000235:genetics*
chemical_list: D016875:tau Proteins