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Exon–intron junctions from a variety of eukaryotes show a high degree of similarity, with the majority of them possessing an invariant GU at the intron's 5′ end and an invariant AG at the intron's 3′ end. Although these highly conserved sequences are required for defining splice junctions, they do not give enough information to do so reliably. Numerous other signals play a role in the splice site definition.

An intron adenosine residue creates a 2′,5′-phosphodiester link with the intron's 5′-terminal phosphate group. As a result, the 5′ exon is freed, and the intron adopts a novel lariat structure (named for its shape). The adenosine at the lariat branch point, which forms three phosphodiester linkages, is usually found 20 to 50 residues upstream of the 3′ splice site in a conserved sequence. Changes to this branch point's mutations At that location, a residue prevents splicing.

The free 3′-OH group of the 5′ exon displaces the 3′ end of the intron, creating a phosphodiester bond with the 3′ exon's 5′-terminal phosphate and resulting in the spliced product. With a free 3′-OH group, the intron is removed in its lariat form. Mutations that change the conserved AG at the 3′ splice junction stop the second step from happening, but they don't stop the lariat from forming. The lariat gradually debranches (linearizes) and degrades quickly in vivo.

This is why splicing is known as transesterification, as it involves these two transesterification reactions.