INTERNAL AU-RICH ELEMENTS MODULATE ACTIVITY OF 2 COMPETING 3'-SPLICE SITES IN PLANT NUCLEI

In vivo analyses using an autonomously replicating Agrobacterium/gemin ivirus vector have enabled identification of AU-rich intronic elements critical for 5' and 3' splice site selection in dicot plant nuclei an d development of a model for pre-mRNA intron recognition in plant nucl ei. To determine the minimal length, spacing and nucleotide compositio ns constraining recognition of the 3' boundary of an intron, two or fo ur nucleotide substitutions have been introduced into the two AU-rich elements located between 50 and 66 nucleotides upstream from the 3' sp lice site of maize Adh1 intron 3. In each case tested, substitutions i n the distal left element (-62 to -66) inactivate the downstream 3' sp lice site at -1 more effectively than substitutions in the proximal ri ght element (-50 to -55). Guanosine or cytosine substitutions in eithe r element reduce recognition of the -1 site significantly; adenosine s ubstitutions 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 wh ich short sets of AU islands bind interactive factors and cooperativel y modulate usage of the downstream splice site. In contrast to the uri dine requirements documented for the 3' terminus of plant introns, ade nosines are partially interchangeable with uridines within this intern al region of the intron.