Internal AU-rich elements modulate activity of two competing 3' splice sites in plant nuclei

In vivo analyses using an autonomously replicating Agrobacterium/geminivirus vector have enabled identification of AU-rich intronic elements critical for 5' and 3' splice site selection in dicot plant nuclei and development of a model for pre-mRNA intron recognition in plant nuclei. To determine the minimal length, spacing and nucleotide compositions constraining recognition of the 3' boundary of an intron, two or four nucleotide substitutions have been introduced into the two AU-rich elements located between 50 and 66 nucleotides upstream from the 3' splice site of maize Adh1 intron 3. In each case tested, substitutions in the distal left element (-62 to -66) inactivate the downstream 3' splice site at -1 more effectively than substitutions in the proximal right element (-50 to -55). Guanosine or cytosine substitutions in either element reduce recognition of the -1 site significantly; adenosine substitutions have a less severe effect. Mutations in both of these AU elements additively block recognition of the downstream 3' splice site. The strong additive effect of these mutations supports a model in which short sets of AU islands bind interactive factors and cooperatively modulate usage of the downstream splice site. In contrast to the uridine requirements documented for the 3' terminus of plant introns, adenosines are partially interchangeable with uridines within this internal region of the intron.