Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency.These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.The human insulin receptor (IR) is encoded by a single gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript. The A isoform lacking exon 11 binds both insulin and IGF-II with high affinity and is expressed ubiquitously; the B isoform containing exon 11 only binds insulin and is expressed predominantly in liver, muscle, adipocytes, and kidney [1,2]. The alternatively spliced exon 11 encodes a 12-amino acid segment (residues 717–728) of the subunit of receptor that disrupts binding to IGF-II [3]. Inclusion of this exon is developmentally and hormonally-regulated and is altered in a number of disease states, such as type II diabetes and myotonic dystrophy, that are associated with insulin resistance [4-10]. The dysregulation of the alternative splicing of the IR may have important consequences for insulin-sensitivity and responsiveness. In previous studies, we have investigated the regulation of splicing of this exon in-vivo using a model minigene system containing exons 10, 11, and 12 and parts of the intervening introns [11]. Recognition of the exon appears to be rate-limiting as partially sp