Use of minigene systems to dissect alternative splicing elements.
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.
Cooper
Thomas A
TA
Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. tcooper@bcm.edu
D000483:Alleles; D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005069:Evaluation Studies as Topic; D005091:Exons; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008967:Molecular Biology / Q000379:methods*; D012322:RNA Precursors / Q000502:physiology; D022821:RNA Splice Sites; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators / Q000502:physiology*; D014162:Transfection
D012322:RNA Precursors; D022821:RNA Splice Sites; D012097:Repressor Proteins; D015534:Trans-Activators