Intron-Mediated Regulation of Plant Gene Expression

Abstract

Introns are transcribed non-coding sequences found in every eukaryote, which are spliced out of the transcript during and following transcription. The position and sequence of an intron can increase mRNA and protein levels in cis by an unexplained ut well-conserved phenomenon termed Intron-Mediated Enhancement (IME). The goal of this thesis was to better understand the IME pathway in plants, by characterising IME-sensitive introns, altering the expression of candidate genes,and screening for novel mutants.

An intron located in the 5’ untranslated region of the Arabidopsis thaliana EF1 gene caused 3 - 25 fold enhancement at both the level of reporter activity and steady-state mRNA in Nicotiana benthamiana, Nicotiana tabaccum and Arabidopsis thaliana, suggesting that enhancement was primarily at the level of steady-state mRNA. Further experiments supported that enhancement was specific to IME rather than transcriptional enhancers, and that enhancement was dependent on a full EF1 promoter in Nicotiana benthamiana and Arabidopsis thaliana, but not Nicotiana tabacum.

The expression of a selection of Nicotiana benthamiana IME candidate gene homologs were transiently modulated by RNA silencing and overexpression. Unexpectedly,both methods increased reporter activity from constructs containing or lacking the EF1 -A2 5’UTR intron, suggesting that IME components may have auto-regulatory feedback, and/or that degradation mechanisms are tightly integrated with IME.

Growth inhibition of Arabidopsis thaliana stably expressing the EF1 promoter and 5’UTR fused to codA, when subjected to the compound 5-fluorocytosine,was dependent on the presence of the 5’UTR intron and thus IME. Chemical mutagenesis yielded ~6000 mutants, three of which lacked growth inhibition that was not due to simple inactivation of the codA gene. These candidates may contain causative mutations that affect the IME pathway.

These results highlighted the complexity of IME in plants, suggesting a tightly controlled IME pathway.