Use of minigene systems to dissect alternative splicing elements

Methods. 2005 Dec;37(4):331-40. doi: 10.1016/j.ymeth.2005.07.015.

Abstract

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.

MeSH terms

  • Alleles
  • Alternative Splicing*
  • Animals
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Enhancer Elements, Genetic / genetics
  • Evaluation Studies as Topic
  • Exons
  • Gene Amplification
  • Gene Expression Regulation*
  • Genes
  • HeLa Cells
  • Humans
  • Introns
  • Molecular Biology / methods*
  • RNA Precursors / physiology
  • RNA Splice Sites*
  • Repressor Proteins / genetics
  • Trans-Activators / physiology*
  • Transfection

Substances

  • RNA Precursors
  • RNA Splice Sites
  • Repressor Proteins
  • Trans-Activators