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Identification of Genetic Interactions Associated with Splicing Factor Prp5

Regulation of gene expression occurs at multiple levels, including the removal of non‐coding sequences from protein‐coding pre‐messenger RNA transcripts, commonly known as pre‐mRNA splicing. Correct gene expression requires high fidelity of pre‐mRNA splicing, as up to 15% of mutations leading to disease may be a result of aberrant pre‐mRNA splicing, including cystic fibrosis, spinal muscular atrophy (SMA) and some leukemias. Pre‐mRNA splicing is a multistep process that requires several proteins and RNAs for ATP hydrolysis, including multiple essential DEAD‐box proteins such as Prp5. Here we investigate the role of the DEAD‐box protein Prp5 in splicing using the yeast Saccharomyces cerevisiae as a model. Prp5 has previously been shown to be required for ATP‐dependent RNA remodeling in splicing, facilitating binding of the U2 snRNA to the branchpoint sequence in the pre‐mRNA intron. In addition, unpublished research has revealed genetic interactions between Prp5 and non‐splicing genes such as Gcn5 and Spt8. In this study, additional potential genetic interactions involving Prp5 were investigated. To identify these interactions, a transposon mutagenesis screen was employed. Approximately 20,000 mutations were screened, 61 of which showed putative genetic suppression of the temperature sensitive prp5‐1 mutation. Subsequent growth assays confirmed that many of these mutations exhibited genetic suppression, indicating important genetic interactions between these genes and Prp5. hiTAIL PCR was utilized to identify a number of these genetic interactions, including an interaction between Prp5 and the MMO1 gene, a verified protein in S. cerevisiae with unknown function. Additional experiments to characterize this interaction with MMO1 are currently being performed. Furthermore, we have used high‐throughput sequencing to identify an additional subset of the interactions from the transposon screen. This study will help shed light upon the role of the DEAD‐box protein Prp5 in pre‐mRNA splicing.