title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro.
abstract: BACKGROUND
  Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Medical Research Service, VASDHS, 3350 La Jolla Village Drive, San Diego, CA 92161, USA. nwebster@ucsd.edu
authors.forename: Nicholas J G
authors.initials: NJ
authors.lastname: Webster
authors.forename: Lui-Guojing
authors.initials: LG
authors.lastname: Evans
authors.forename: Matt
authors.initials: M
authors.lastname: Caples
authors.forename: Laura
authors.initials: L
authors.lastname: Erker
authors.forename: Shern L
authors.initials: SL
authors.lastname: Chew
mesh_terms: D017398:Alternative Splicing; D045744:Cell Line, Tumor; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D017354:Point Mutation; D020033:Protein Isoforms; D022821:RNA Splice Sites; D011972:Receptor, Insulin / Q000235:genetics*; D016914:Ribonuclease H; D013698:Templates, Genetic
chemical_list: D020033:Protein Isoforms; D022821:RNA Splice Sites; D011972:Receptor, Insulin; D016914:Ribonuclease H