Intron‐mediated enhancement of transgene expression in maize is a nuclear, gene‐dependent process

Abstract

SummaryIn monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5' untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5' UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron-mediated enhancement of expression (IME) is gene-dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability, it may mean that IME rescues rather than stimulates gene expression.

Promoter-Proximal Introns in Arabidopsis thaliana Are Enriched in Dispersed Signals that Elevate Gene Expression

“…Some efficiently spliced introns boost expression more than 10-fold, while others have little or no effect, arguing against generic mechanisms related to splicing that would apply to all introns equally. Introns enhance gene expression by increasing the steady state amount of mature mRNA in the cell (Callis et al, 1987;Dean et al, 1989;Rethmeier et al, 1997;Rose and Last, 1997;Nott et al, 2003), apparently without significantly changing mRNA stability (Rethmeier et al, 1997;Nott et al, 2003). Although modest effects of introns on transcription have been noted (Rose and Last, 1997;Fong and Zhou, 2001;Furger et al, 2002), these are insufficient to entirely explain the increase in mRNA accumulation.…”

Requirements for intron-mediated enhancement of gene expression in Arabidopsis

“…The frequently observed stimulation of expression caused by plant introns has been termed intronmediated enhancement (IME) (Mascarenhas et al+, 1990)+ The difference in amount of product derived from an intron-containing gene and an otherwise identical intronless construct is typically between 2-and 10-fold but can be significantly more, especially in monocots (Maas et al+, 1991;Zhang et al+, 1994)+ The magnitude of the enhancement varies depending on the promoter, intron, and reporter gene used (Callis et al+, 1987;Luehrsen & Walbot, 1991;Rethmeier et al+, 1998), the sequences that flank the intron (Maas et al+, 1991;Clancy et al+, 1994), and the type of cell or tissue in which expression is determined (Gallie & Young, 1994)+ Even though the high degree of variability makes comparisons between studies difficult, and introns could affect expression in different ways, some common themes have emerged that help to delineate potential mechanisms of IME+ The increase in expression mediated by introns is usually apparent at the level of mRNA accumulation (Callis et al+, 1987;Dean et al+, 1989;Rethmeier et al+, 1997;Rose & Last, 1997;Wang et al+, 2002)+ However, IME is unlike the enhancement caused by transcriptional enhancer elements in that the introns must be located within transcribed sequences and in their normal orientation to boost expression (Callis et al+, 1987;Mascarenhas et al+, 1990;Clancy et al+, 1994)+ The distinction from transcriptional enhancer elements is further supported by the demonstration that the introns tested increase mRNA accumulation without significantly affecting the rate of transcript initiation, as de-termined by nuclear run-on transcription assays (Dean et al+, 1989;Rose & Last, 1997;Rose & Beliakoff, 2000)+ Although introns could elevate steady-state mRNA levels by increasing mRNA stability, the half-life of mRNA from genes with or without an intron has been reported to be the same (Nash & Walbot, 1992;Rethmeier et al+, 1997)+ Thus, the mechanism of IME remains unclear, but presumably operates at a co-or posttranscriptional level+ It is plausible that the enhancement mediated by introns is functionally connected to their recognition and removal by the splicing machinery+ Thus, the features that identify a sequence as an intron and/or participate in spliceosome assembly and the splicing reactions might also be involved in IME+ In Arabidopsis, the only recognizable characteristics of introns are that they have weakly ...…”

Splicing of the Maize Sh1 First Intron Is Essential for Enhancement of Gene Expression, and a T-Rich Motif Increases Expression without Affecting Splicing

“…Plant introns that stimulate expression have been documented in petunia (Petunia hybrida; Dean et al, 1989;Vain et al, 1996), oat (Avena sativa; Bruce and Quail, 1990), rice (Oryza sativa; McElroy et al, 1990;Snowden et al, 1996;Rethmeier et al, 1997), castor bean (Ricinus communis; Tanaka et al, 1990), potato (Solanum tuberosum; Leon et al, 1991;Fu et al, 1995aFu et al, , 1995b, Arabidopsis (Rose and Last, 1997;ChaubetGigot et al, 2001), soybean (Glycine max; Kato et al, 1998), and tobacco (Nicotiana tabacum; Plesse et al, 2001). The approximately 2-to 10-fold range of intron-mediated enhancement usually seen in dicots is much less than increases observed in monocots, which can be more than 100-fold (for review, see Simpson and Filipowicz, 1996).…”

“…The degree of enhancement is usually greater for relatively weak promoters (Callis et al, 1987;Vasil et el., 1987;Mascarenhas et al, 1990;. The magnitude of stimulation also depends on the coding sequences (Sinibaldi and Mettler, 1992;Rethmeier et al, 1997Rethmeier et al, , 1998, the tissue of expression, and physiological conditions (Tanaka et al, 1990;Sinibaldi and Mettler, 1992;Gallie and Young, 1994;Park, 1995a, 1995b;ChaubetGigot et al, 2001;Plesse et al, 2001).…”