Expression of maize Adh1 intron mutants in tobacco nuclei

Abstract

In vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. Differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. To locate elements which modulate efficient recognition of introns in dicot nuclei, the maize Adh1 gene has been expressed in full-length and single intron constructs in Nicotiana benthamiana nuclei using an autonomously replicating plant expression vector. Quantitative PCR-Southern analyses indicate that the inefficient splicing of the maize Adh1 intron 1 (57% AU) in these dicot nuclei can be dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5' splice site. This indicates that the 5' splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei and that, most importantly, the remainder of this monocot intron contains no elements which inhibit its accurate recognition in dicot nuclei. Deletions in intron 3 (66% AU) which effectively move the 3' boundary between AU-rich intron and GC-rich exon sequences strongly activate a cryptic upstream splice site; those which do not reposition this boundary activate a downstream cryptic splice site. This suggests that 3' splice site selection in dicot nuclei is extremely flexible and not dependent on strict sequence requirements but rather on the transition points between introns and exons. Our results are consistent with a model in which potential splice sites are selected if they are located upstream (5' splice site) or downstream (3' splice site) of AU transition points and not if they are embedded within AU-rich sequences.