Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia.
Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.
Hims
Matthew M
MM
Center for Human Genetic Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Ibrahim
El Chérif
EC
Leyne
Maire
M
Mull
James
J
Liu
Lijuan
L
Lazaro
Conxi
C
Shetty
Ranjit S
RS
Gill
Sandra
S
Gusella
James F
JF
Reed
Robin
R
Slaugenhaupt
Susan A
SA
D002352:Carrier Proteins / Q000032:analysis / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D004402:Dysautonomia, Familial / Q000188:drug therapy*; D005091:Exons / Q000187:drug effects; D006801:Humans; D007701:Kinetin / Q000494:pharmacology / Q000627:therapeutic use*; D017253:Neurofibromatoses / Q000188:drug therapy / Q000235:genetics; D012326:RNA Splicing / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects; D035602:Transcriptional Elongation Factors
D002352:Carrier Proteins; C114720:Elp1 protein, human; D012333:RNA, Messenger; D035602:Transcriptional Elongation Factors; D007701:Kinetin