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

Abstract

Certain plant and animal introns increase expression of protein-coding sequences when placed in the 5Ј region of the transcription unit. The mechanisms of intron-mediated enhancement have not been defined, but are generally accepted to be post-or cotranscriptional in character. One of the most effective plant introns in stimulating gene expression is the 1,028-bp first intron of the Sh1 gene that encodes maize (Zea mays) sucrose synthase. To address the mechanisms of intron-mediated enhancement, we used reporter gene fusions to identify features of the Sh1 first intron required for enhancement in cultured maize cells. A 145-bp derivative conferred approximately the same 20-to 50-fold stimulation typical for the full-length intron in this transient expression system. A 35-bp motif contained within the intron is required for maximum levels of enhancement but not for efficient transcript splicing. The important feature of this redundant 35-bp motif is T-richness rather than the specific sequence. When transcript splicing was abolished by mutations at the intron borders, enhancement was reduced to about 2-fold. The requirement of splicing for enhancement was not because of upstream translation initiation codons contained in unspliced transcripts. On the basis of our current findings, we conclude that splicing of the Sh1 intron is integral to enhancement, and we hypothesize that transcript modifications triggered by the T-rich motif and splicing may link the mRNA with the trafficking system of the cell.Most plant genes contain intervening sequences (introns) that are transcribed into pre-mRNA and later removed by splicing. Introns separate gene segments (exons) that hold protein-coding information or are non-coding but retained in the mature transcript. The observation that some introns stimulate gene expression was first made in animal systems and extended to plants when Callis et al. (1987) demonstrated that the maize (Zea mays) Adh1 first intron increased expression of several genes. Other maize introns that have been shown to increase expression include the Bz1 intron (Callis et al., 1987), Hsp82 first intron (Silva et al., 1988), Sh1 first intron (Vasil et al., 1989), Adh1 introns 2 and 6 (Mascarenhas et al., 1990), actin third intron (Luehrsen and Walbot, 1991), GapA1 first intron (Donath et al., 1995), Ubi1 first intron (Vain et al., 1996), and the RpoT fourth intron (Bourdon et al., 2001).Plant introns that stimulate expression have been documented in petunia (Petunia hybrida; Dean et al., 1989;Vain et al., 1996) 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). Not all plant introns enhance gene expression. Three dicot introns do no...

Dissection of Maize Kernel Composition and Starch Production by Candidate Gene Association

“…This has suggested several specific biochemical hypotheses that should be tested in the future: (1) a sh1 polymorphism located in intron one associated with a G 3 E effect-this intron has been related to gene activity (Vasil et al, 1989;Clancy et al, 1994;Clancy and Hannah, 2002). (2) Allele Bt2-925(T), located in bt2's exon one and causing a P22L polymorphism, associated with a decrease in variance in oil content.…”

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

“…For example, removing 883 nucleotides from the 1028-nucleotide first intron of the maize (Zea mays) Sh1 gene has a negligible effect on its enhancing ability (Clancy and Hannah, 2002), and all sequences in Arabidopsis thaliana TRP1 intron 1 can be individually deleted without reducing the stimulation it mediates (Rose and Beliakoff, 2000). The sequences responsible for increasing expression are therefore likely to be redundant and dispersed throughout enhancing introns.…”

Cloning of SCA Gene Related to Pollen Tube Adhesion and Oriented Growth and Analysis of Gene Diversity in <i>Lilium spp.</i>

“…Although intron does not encode protein, it plays an important regulatory role in gene expression. In most of cases, intron will enhance gene expression (Callis et al, 1987), of which the enhanced degree of gene expression would be related with many factors, such as characteristics of intron (Clancy and Hannah, 2002), features of promoter and exon sequence, intron positions on the vector (Wang, 2008), the sequence structure of the target gene, cell type and cell physiological status. Some introns have tissue-specific regulation to gene expression (Stemmler et al, 2005), while some introns have a disincentive function on gene expression (Hormuzdi et al, 1998).…”