To explore possible mechanisms of intron-mediated enhancement
of gene expression, the features of PAT1 intron 1 required
to elevate mRNA accumulation were systematically tested in
transgenic Arabidopsis. This intron is remarkably
resilient, retaining some ability to increase mRNA accumulation
when splicing was prevented by mutation of 5′ and 3′
splice sites, branchpoint sequences, or when intron U-richness
was reduced. Enhancement was abolished by simultaneously
eliminating branchpoints and the 5′ splice site, structures
involved in the first two steps of spliceosome assembly. Although
this suggests that the splicing machinery is required, intron
splicing is clearly not enough to enhance mRNA accumulation.
Five other introns were all efficiently spliced but varied widely
in their ability to increase mRNA levels. Furthermore,
PAT1 intron 1 was spliced but lost the ability to elevate
mRNA accumulation when moved to the 3′ UTR. These findings
demonstrate that splicing per se is neither necessary nor
sufficient for an intron to enhance mRNA accumulation, and suggest
a mechanism that requires intron recognition by the splicing
machinery but also involves nonconserved intron sequences.