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SRp20, hnRNP A1 and F regulate alternative splicing of insulin receptor via binding to the intronic regulatory elements
Author(s) -
Talukdar Indrani,
Sen Supriya,
Webster Nicholas
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.601.1
Subject(s) - minigene , exon , microbiology and biotechnology , biology , intron , rna splicing , alternative splicing , rna binding protein , mutant , transfection , rna , genetics , gene
Insulin receptor has two splice variants which are produced due to alternative splicing of 36 base pair long exon 11. Inclusion and exclusion of this exon in the matured RNA produces two isoforms, B and A respectively. Previously our lab has generated a minigene construct containing exon 10–12 and the surrounding introns of human IR. Deletion mutation of this construct identified a purine rich ISE near the 5′ and an ISS near the 3′ end of intron 10. For accurate identification of these elements linker scanning mutants were created throughout the ISE and ISS. These constructs were transfected into HepG2 cell lines. The ISE mutants suggest the presence of multiple element binding sites in this region. From ISS mutants, a 12 nt long silencer sequence has been identified, deletion or mutation of which caused a constitutive incorporation of exon 11. Sequence analysis of ISE revealed that it contains potential binding sites for hnRNP A1, F and H proteins. An in vitro RNA pull down assay taking portions of the ISE sequence followed by western blot analysis confirms the binding of these proteins to this region. Co transfection of the minigene with hnRNP A1 inhibits and hnRNP F activates the inclusion of exon 11 in HepG2 cells. Co transfection of the minigene with siRNA against hnRNPA1 and F increases and decreases the inclusion of exon 11 respectively. RNA pull down assay with the ISS revealed the binding of CUG‐binding protein 1 (CUGBP‐1) and SRp20. Co transfection of wild type minigene with CUGBP‐1 inhibits the inclusion of exon 11, whereas, mutation of the 12‐nt region of the minigene abolishes the effect of CUGBP‐1. Surprisingly, co‐transfection of the minigene with SRp20 increased the inclusion of exon 11, suggesting that SRp20 might bind to an enhancer element as well. This study identifies novel intronic regulatory elements and reveals the role of hnRNP A1, F and SRp20 as regulatory proteins for IR splicing.