Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression.

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism has not been elucidated yet, but gene expression regulation is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm, called IMEter, able to predict the IME potential of introns without direct testing has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was experimentally tested only on the first plant species, by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, we used a vector bearing the upstream regulatory sequence of a rice beta tubulin gene (OsTub6) fused to the reporter GUS gene. The enhancing intron interrupting the OsTub6 5’UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli was shown to correlate to the calculated IMEter scores. We also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5’ region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.