In potato invertase genes, the constitutively included, 9-nucleotide (nt)-long mini-exon requires a strong branchpoint and U-rich polypyrimidine tract for inclusion. The strength of these splicing signals was demonstrated by greatly enhanced splicing of a poorly spliced intron and by their ability to support splicing of an artificial mini-exon, following their introduction. Plant introns also require a second splicing signal, UA-rich intronic elements, for efficient intron splicing. Mutation of the branchpoint caused loss of mini-exon inclusion without loss of splicing enhancement, showing that the same U-rich sequence can function as either a polypyrimidine tract or a UA-rich intronic element. The distinction between the splicing signals depended on intron context (the presence or absence of an upstream, adjacent and functional branchpoint), and on the sequence context of the U-rich elements. Polypyrimidine tracts tolerated C residues while UA-rich intronic elements tolerated As. Thus, in plant introns, U-rich splicing elements can have dual roles as either a general plant U-rich splicing signal or a polypyrimidine tract. Finally, overexpression of two different U-rich binding proteins enhanced intron recognition significantly. These results highlight the importance of co-operation between splicing signals, the importance of other nucleotides within U-rich elements for optimal binding of competing splicing factors and effects on splicing efficiency of U-rich binding proteins.