A Splicing Reporter Tuned to Non-AG Acceptor Sites Reveals that Luteolin Enhances the Recognition of Non-canonical Acceptor Sites.
Removal of an intron requires precise recognition of the splice donor and acceptor sites located at the 5' and 3' termini of introns. Although the roles of these sequences differ, mutations in both sites easily block normal splicing and produce an aberrant mRNA. For example, many splice-site mutations occur in patients with inherited diseases. Several approaches have been evaluated to restore expression of a functional protein; however, because of the strict requirement for an AG dinucleotide at the 3' terminus of a U2-type intron, no method is available to correct splicing at a mutated sequence. To identify compounds that allow splicing at the non-AG acceptor site, in the present study we constructed a reporter gene with a modified polypyrimidine tract. However, the modified polypyrimidine tract mediated splicing at adjacent non-canonical acceptor sites, including the original mutated site. Further, we show that certain flavones such as luteolin and apigenin enhanced aberrant splicing at the non-canonical acceptor site of the reporter gene. These results suggest that the reporter gene and luteolin may be useful for further screening to identify molecules that correct aberrant splicing caused by a disease-associated splice acceptor site mutation.
Graduate School of Life Science, Hokkaido University, Sapporo, 060-0812, Japan.
Masanori
M
Chiba
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
Hiroyoshi
H
Ariga
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
Hiroshi
http://orcid.org/0000-0002-9620-4651
H
Maita
D001483:Base Sequence; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D047311:Luteolin / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D022821:RNA Splice Sites; D020133:Reverse Transcriptase Polymerase Chain Reaction
D022821:RNA Splice Sites; D012313:RNA; D047311:Luteolin
3′ splice site; fidelity; luteolin; pre-mRNA splicing; reporter assay