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

BMC Molecular Biology
Nicholas J G WebsterShern L Chew

Abstract

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. 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.

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Citations

Dec 3, 2008·Molecular and Cellular Biology·Supriya SenNicholas J G Webster
May 10, 2008·Archives of Physiology and Biochemistry·Francesco FrascaRiccardo Vigneri
Aug 9, 2019·The Journal of Clinical Investigation·Deepak KumarNicholas Jg Webster

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Methods Mentioned

BETA
PCR
transfection
RNaseH
electrophoresis
in-vitro
in-vitro transcription
transfections

Software Mentioned

Kodak 1D

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