Figure 1. Splicing reporter vectors. (a) An intronless zein gene from maize was cloned between the CaMV promoter and terminator sequences (shaded boxes), and a unique BamHI restriction site was introduced into the coding sequence (white box) to produce the plasmid, pDH515. Introns of interest and surrounding exon sequence (solid line separating the dark boxes) were introduced in frame into the BamHI site. The lines indicate the intron removed by splicing; (b) The potato invertase mini‐exon was introduced into pDH515. The mini‐exon is 9 nt long and requires strong splicing signals in the upstream intron to be efficiently spliced. This construct formed the basis of a series of intron signal mutations; (c) For a visible assay of splicing, the invertase mini‐exon system was modified to be expressed as a fusion protein to GFP. The introduction of a stop codon into the mini‐exon produced GFP fluorescence only when the mini‐exon was skipped. This construct, and derivatives of it, have been used in agroinfiltration experiments along with protein factors.

Figure 2. Splicing of U12 and U2 introns using tobacco protoplast transfection. (a) Construct pGSH9 consisting of GSH2 U12 intron with both upstream and downstream U2 introns inserted within the coding sequence of the maize zein gene. The lines indicate intron sequences, while boxes indicate exons. The zein gene coding sequence is labeled, and all possible splicing events are indicated by the lines across the introns. Arrows indicate the primer positions used in the RT‐PCR analysis; (b) Genescan analysis of splicing of pGSH9 construct alone and with overexpressed RBP45 and UBP1 proteins in tobacco protoplasts. M indicates a DNA size marker in base pairs. The splicing of pGSH9 gave three different RT‐PCR products, in addition to unspliced transcripts where either the downstream U2 intron was removed (D) or where both U2 introns were removed (UD) and the fully spliced product (FS). Overexpression of UBP1 did not alter the splicing pattern of pGSH9, but overexpression of RBP45 caused exon skipping (SK); (c) Protein gel blot analysis with anti‐HA antibody to HA‐tagged RBP45 and UBP1 proteins overexpressed in tobacco protoplasts. The protein bands are indicated by asterisks. C represents control, untransfected protoplasts.