Use of minigene systems to dissect alternative splicing elements

TA Cooper - Methods, 2005 - Elsevier
Methods, 2005Elsevier
Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The
splicing efficiency of individual exons is determined by multiple features involving gene
architecture, a variety of cis-acting elements within the exons and flanking introns, and
interactions with components of the basal splicing machinery (called the spliceosome) and
auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both
alternative and constitutive exons are recognized by multiple weak protein: RNA interactions …
Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.
Elsevier