Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

Abstract

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression. Although many genetic tools and basic transformation strategies exist for the model microalga Chlamydomonas reinhardtii, high-level genetic engineering with this organism is hindered by its inherent recalcitrance to foreign gene expression and limited knowledge of responsible expression regulators. In this work, we characterized the dynamics of 33 endogenous and 13 non-native introns and their effect on gene expression as artificial insertions into codon optimized transgenes. We found that introns from different origins have the capacity to increase gene expression rates. Intron-mediated enhancement was observed exclusively when these elements were placed in transcripts but not outside of ORFs. Insertion of different endogenous introns into coding sequences was found to positively affect expression rates through a synergy of additive transcription enhancement and exon length reduction, similar to those natively found in the C. reinhardtii genome. Our results indicate that intensive mRNA processing plays an underestimated role in the regulation of native gene expression in C. reinhardtii. In addition to internal sequence motifs, the localization of artificially introduced introns greatly affected transgene expression levels. This work is highly valuable to the greater microalgal and synthetic biology research communities and contributes to broadening our understanding of eukaryotic intron-mediated enhancement.