3' SPLICE-SITE SELECTION IN DICOT PLANT NUCLEI IS POSITION-DEPENDENT

In contrast to mammalian and yeast systems, the mechanism for intron r ecognition and splice site selection in plant pre-mRNAs is poorly unde rstood. Splice site sequences and putative branchpoint sequences are l oosely conserved in plant introns compared with other eukaryotes. Perh aps to compensate for these variations, plant introns are significantl y richer in adenosine and uridine residues than are their adjacent exo ns. To define elements critical for 3' splice site selection in dicoty ledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by usi ng an autonomously replicating plant expression vector. Using a series of intron rearrangements which reposition the 3' intron-exon border, we demonstrate that the normal 3' splice site is defined in a position -dependent manner and that cryptic 3' splice sites within the intron a re masked by the presence of a functional downstream 3' splice site. D isruption of the AU-rich elements upstream from the normal 3' splice s ite indicates that multiple AU elements between -66 and -6 cooperative ly define the 3' boundary of the intron. These results are consistent with a model for plant intron recognition in which AU-rich elements sp read throughout the length of the intron roughly define the intron bou ndaries by generating strong AU transition points. Functional 3' splic e sites located downstream from these AU-rich sequences are preferenti ally selected over sites embedded within them.