The alternative splicing (AS) of precursor messengerRNA(pre‐mRNA) is a tightly regulated process through which introns are removed to leave the resulting exons in themRNAappropriately aligned and ligated. TheASof pre‐mRNAis a key mechanism for increasing the complexity of proteins encoded in the genome. In humans, more than 90% of genes undergoAS, underscoring the importance of this process inRNAbiogenesis. As such,ASmisregulation underlies multiple human diseases. The splicing reaction is catalyzed by the spliceosome, a highly dynamic complex that assembles at or near the intron/exon boundaries and undergoes sequential conformational and compositional changes during splicing. The initial recognition of splice sites defines the exons that are going to be removed, which is a critical step in the highly regulated splicing process. Although the available lines of evidence are increasing, the molecular mechanisms governingAS, including the initial interactions occurring at intron/exon boundaries, and the factors that modulate these critical connections by functioning as a scaffold for active‐siteRNAsor proteins, remain poorly understood. In this review, we summarize the major hallmarks of the initial steps in the splicing process and the role of auxiliary factors that contribute to the assembly of the spliceosomal complex. We also discuss the role of the essential yeast Prp40 protein and its mammalian homologs in the specificity of this pre‐mRNAprocessing event. In addition, we provide the first exhaustive phylogenetic analysis of the molecular evolution of Prp40 family members.WIREs RNA2016, 7:17–32. doi: 10.1002/wrna.1312This article is categorized under:RNA Processing > Splicing MechanismsRNA Processing > Splicing Regulation/Alternative Splicing