Yeast Pre-mRNA splicing: Methods, mechanisms, and machinery

Abstract

Publisher Summary This chapter discusses the process of pre-mRNA splicing in yeasts. The chapter outlines the history of pre-mRNA splicing. The genes for dozens of protein factors involved in the removal of pre-mRNA introns have been noted in the deeply rooted eukaryote Giardia lamblia , indicating that introns are present from the simpler eukaryotes to humans. Yeasts have only a few hundred introns, generally limited to one intron per intron-containing gene. For successful splicing, intronic sequences must be defined precisely to preserve the reading frame. The chapter presents a schematic of the products of each of the chemical steps of the splicing reaction. Although the signals are more divergent in humans, the chemistry of the pre-mRNA splicing reaction is conserved through evolution. The products of the first step of splicing are the 5' exon and the lariat intermediate, which contains the intron in the form of the branched lariat. The second step of splicing produces the mature mRNA and the lariat intron. The chapter discusses in vitro splicing extract preparation from Saccharomyces cerevisiae ( S.cerevisiae ) along with in vivo splicing reporter assays and native gel analysis of splicing complexes. The chapter also discusses various affinity purification techniques.