Engineering splicing factors with designed specificities.
Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.
Wang
Yang
Y
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Cheong
Cheom-Gil
CG
Hall
Traci M Tanaka
TM
Wang
Zefeng
Z
D017398:Alternative Splicing / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001698:Biomedical Engineering / Q000379:methods*; D045744:Cell Line, Tumor; D005091:Exons / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D051020:bcl-X Protein / Q000235:genetics*
D000970:Antineoplastic Agents; D020033:Protein Isoforms; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D051020:bcl-X Protein; C465505:pumilio protein, human