3' Splice Site Selection in Dicot Plant Nuclei Is Position Dependent

In contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mRNAs is poorly understood. Splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes. Perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons. To define elements critical for 3' splice site selection in dicotyledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by using 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 are masked by the presence of a functional downstream 3' splice site. Disruption of the AU-rich elements upstream from the normal 3' splice site indicates that multiple AU elements between -66 and -6 cooperatively define the 3' boundary of the intron. These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU transition points. Functional 3' splice sites located downstream from these AU-rich sequences are preferentially selected over sites embedded within them.