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.

Synthetic biology is essential to unlock commercial biofuel production through hyper lipid-producing microalgae: a review

“…The DBTL cycle is becoming increasingly relevant due to advancements in affordable and high-throughput gene synthesis (Kosuri et al, 2010;Rogers & Church, 2016) and the development of efficient gene assembly, such as Golden Gate cloning, and the related standardized Modular Cloning (MoClo) toolkit, that enable one pot and one-step synthesis of multi-gene constructs from standardized constituent parts (Engler et al, 2008;Crozet et al, 2018). Also, rapid advancements in mutagenic and transgenic efficiencies (Ferenczi, Pyott, Xipnitou, & Molnar, 2017;Serif et al, 2018;Angstenberger et al, 2018;Picariello et al, 2020), increasing understanding of regulatory elements (Baier, Jacobebbinghaus, Einhaus, Lauersen, & Kruse, 2020;Mehrshahi et al, 2020;Rose, 2019), coupled with high-performance screening techniques enabled by optimization of microfluidics (Nouemssi et al, 2020;Saad et al, 2019;Südfeld, Hubáček, D'Adamo, Wijffels, & Barbosa, 2020;Wheeler et al, 2003), have cumulatively enabled the automation of DBTL workflow (Carbonell et al, 2018). Most notably, the integration of machine learning has exponentially increased productivity in recent years (Carbonell, Le Feuvre, Takano, & Scrutton, 2020;Opgenorth et al, 2019;Radivojević, Costello, Workman, & Garcia Martin, 2020).…”

The Role of Introns for the Development of Inflammation-Mediated Cancer Cell

“…After the elucidation of intron splicing mechanism, scientist became excited about its function on gene expression and speculated that may be introns carry out some function like regulation of splicing function, regulation of transcription, evolutionary function or coding capacity but there was no clear examples of their active functions on gene expression [41]. From the starting 21st century, many researches have claimed about the intron function on gene expression or intron mediated enhancement of gene expression [13,[42][43][44][45][46][47]. However, a question remains unclear that within the genome, who is responsible to remember or decide which intron or parts of nucleotide sequences are necessary to stay within the mature mRNA stand for inflammatory gene expression rather than form splicing that result in cancer?…”

Gene Architecture Facilitates Intron-Mediated Enhancement of Transcription

“…Soon thereafter it was realized that introns play a general role in activating transcription in a variety of eukaryotes. Intronmediated regulation of transcription has been observed in simple eukaryotes like yeast and Chlamydomonas as well as in higher eukaryotes like flies, worms, plants, and mammals including humans (Callis et al, 1987;Brinster et al, 1988;Bieberstein et al, 2012;Moabbi et al, 2012;Agarwal and Ansari, 2016;Baier et al, 2020). This enhancement property could be a very crucial function of introns in eukaryotes as a number of genes are dependent on introns for their normal transcription (Rose, 2019).…”

“…This enhancement property could be a very crucial function of introns in eukaryotes as a number of genes are dependent on introns for their normal transcription (Rose, 2019). In general, intron-containing genes in eukaryotes exhibit higher expression than their non-intronic counterparts (Ares et al, 1999;Juneau et al, 2006;Bieberstein et al, 2012;Gallegos and Rose, 2015;Ding and Elowitz, 2019;Baier et al, 2020). An intron may enhance transcription by a meager 2-3-fold, or it may augment mRNA output in the range of 10-100-fold or higher depending on the gene.…”