A splicing‐competent intron has been found to affect the expression of a gene at almost every step between transcription and translation. In this investigation, we performed a comprehensive analysis of the role of an intron from two yeast genes, ASC1 and APE2 , on cellular fitness and at different steps of gene expression. Mutants containing intronless versions of either gene grew 30–60% slower than their intron‐containing counterparts, and their growth was compromised on ethanol and glycerol medium. Furthermore, …