Advanced Methods for the Analysis of Altered Pre-mRNA Splicing in Yeast and Disease

Splicing of pre-messenger RNA (pre-mRNA) transcripts is a fundamental process in all eukaryotes that provides a mechanism of increasing the proteomic diversity within a cell that can be tightly regulated in a dynamic manner. While constitutive and alternative splicing are necessary for the correct development and regulation of cells/organisms, aberrant splicing is now associated with an increasingly varied number of human diseases, such as neurological and developmental diseases, and cancer. Studies of splicing mechanisms and regulation are often achieved in nonhuman model organisms such as yeast. Yeasts possess homologs to many of the core spliceosome components of higher organisms, including humans, and as such yeast species are now a well-established model organism for understanding how differential splicing of transcripts can alter the phenotype of a cell or organism. Here we describe methods to investigate pre-mRNA splicing in yeast cells using modern RNA-Seq technology and bioinformatics software. Details of traditional validation methods are also described.