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

Abstract

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression 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, which is 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 only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice β-tubulin gene (OsTub6) fused to the GUS reporter gene was used. 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 does not significantly correlate with the calculated IMEter score. It was 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.