U-rich tracts enhance 3′ splice site recognition in plant nuclei

The process of 5′ and 3′ splice site definition in plant pre-mREMA splicing differs from that in mammals and yeast. In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5′ splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3′ splice site; in plants, the 3′ intron boundary is defined in a position-dependent manner relative to AU-rich elements within the intron. To determine if uridines are utilized to any extent in plant 3′ splice site recognition, uridines in the region preceding the normal (-1) 3′ splice site of pea rbcS3A intron 1 were replaced with adenosines. This mutant activates two cryptic 3′ splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (-1) site are essential for recognition. Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3′ splice site containing 14 or 10 uridine tracts with a G at -4 can effectively outcompete the normal 3′ splice site containing an eight uridine tract with a U at -4. Substitutions at the -4 position demonstrated that the identity of the nucleotide at this position greatly affects 3′ splice site selection. It has been concluded that several factors affect competition between these 3′ splice sites. These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3′ terminal CAG/ and the identity of nucleotide -4.