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 5' 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.

Figure 1

Diagrams of chimeric gene constructions for transient expression. A, Construct 35SIfSCN contains the 1,028-bp Sh1 first intron flanked by short portions of Sh1 exons 1 and 2 (E1 and E2). The 35S promoter is from CaMV, CAT encodes chloramphenicol acetyltransferase, and the termination region is the 3′ part of the nopaline synthase gene. The transcription start site is designated by the arrow, and the CAT translation initiation codon is indicated as ATG. B, The control construct 35SECN lacks the Sh1 first intron but is otherwise identical to construct 35SIfSCN. C, Deletions of internal portions of the Sh1 first intron were made while retaining all other elements of construct 35SIfSCN. Diagrams of the deletion derivatives indicate the basepairs retained from the 5′ and 3′ ends of the intron.

Figure 2

Mutation of Sh1 first intron termini abolishes transcript splicing. Total RNA (1.0 μg) from cultured cells transfected with each construct was amplified by RT-PCR. The products were analyzed by gel-blot hybridization using a ³²P-labeled bp probe composed of 5′-UTR and CAT sequences. The 230-bp probe is identical to the RT-PCR products from spliced transcripts.