A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

Abstract

Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

Introns as Gene Regulators: A Brick on the Accelerator

“…None of the genes identified in the same study that are highly expressed only in certain tissues contained an intron with a high IMEter score. While the role of introns in the expression of these genes has been only partially investigated (Zhang et al, 2016), these results suggest not only that introns are involved in activating the most strongly and widely expressed genes in the genome, but also that the intron may have a larger role than the promoter in driving this high level of expression.…”

“…This is in agreement with the kinds of genes in which stimulating introns historically have been discovered by comparing the expression of cDNA and genomic versions of the same gene. While this represents a small sample rather than an exhaustive survey, and there are multiple exceptions, many of the genes that contain a stimulating intron encode proteins that are needed in large amounts in most cell types, such as ubiquitin (Norris et al, 1993; Plesse et al, 2001), actin (Weise et al, 2006; Jeong et al, 2009), tubulin (Jeon et al, 2000; Fiume et al, 2004), ribosomal proteins (Chung and Perry, 1989; Ares et al, 1999), or elongation factors (Curie et al, 1991; Zhang et al, 2016). Additional evidence that introns generally produce strong constitutive gene expression is that inserting an expression-stimulating intron into a gene that is normally active only in certain cell types can override the regulation provided by the promoter and result in widespread expression (Jeong et al, 2006; Emami et al, 2013).…”

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters

“…Introns, particularly the first intron in the 5 untranslated region (5 UTR), via intron mediated enhancement (IME) can significantly increase gene expression. From literature review, we selected the intron sequence from soybean elongation factor 1A (eEF1A) gene (GmScreamM8) that was reported for high activity in native promoter [82]. The sequence of synthetic intron part 3 (4xEF4-M8CIN3) 222 bp showed higher expression than other parts.…”

“…Six vector constructs were designed specifically for GUS expression in transient expression system of soybean transgenic hairy roots. Synthetic modules named SynP14, SynP15, Syn P16, SynP17, SynP18, and SynP19 were constructed by fusing the different sets of multiple cis motifs and a root motif with intron sequence from soybean elongation factor 1A (EF1A) gene (third intron 222 bp) [82] upstream of 35S minimal promoter. For synthetic module SynP14, multiple copies of cis motifs and synthetic intron sequence were fused upstream of 35S minimal promoter sequences and in SynP15, the same elements were fused at both the 5 and 3 ends of minimal promoter.…”

Changes to the core and flanking sequences of G‐box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters

“…Briefly, upper and lower phosphorylated oligonucleotides of each element‐containing sequence were first annealed together to generate a monomer with Spe I and Xba I overhangs at the 5′ and 3′ ends respectively. The annealed fragment was then transcriptionally fused to the green fluorescent protein ( gfp ) gene in the 35Score‐pFLEV (GenBank accession number: ) or GmScreamM8C‐pFLEV plasmids (GenBank accession number: ) (Zhang et al ., ). The 35Score‐pFLEV or GmScreamM8C‐pFLEV constructs contain a gfp gene regulated by the 46‐bp 35S core promoter or the 100‐bp GmScreamM8 core promoter with a native GmScreamM8 leader intron respectively.…”