4702 Nucleic Acids Research, 2012, Vol. 40, No. 10 Figure 1. The P5SM splicing cassette functions in the eGFP coding sequence. (A) Schematic representation of the P5SM-splicing cassette (boxed) within gene constructs TFIIIA-eGFP (native context, top) and eGFP-P5SME/R (foreign context, bottom). The full sequence maps for these constructs are shown in Supplementary Figure S6. The eGFP coding sequence is shaded. The cassette exon contains the P5SM RNA element and an in-frame premature termination codon (X). The splicing reactions that generate spliced products SP-I and SP-II are shown. (B) eGFP-P5SME/R spliced products detected by RT–PCR upon induction with AtL5 (+) or LUC as a control ( ). (C) Relative amounts of the spliced products of eGFP-P5SME/R determined by RT–qPCR. Data are averaged with number of biological replicates (n) and standard deviations shown. The p-value was determined using the paired t-test. (D) Fold induction of protein expression quantiﬁed by eGFP ﬂuorescence for each construct. Induction with AtL5 was measured in comparison to induction with LUC as a control. (E) Representative whole leaf scan of eGFP ﬂuorescence 3 days post-inﬁltration for each construct. The right leaf halves coexpressed AtL5 and the left halves coexpressed LUC (control). for nonsense-mediated decay (NMD) instead of undergo- ing translation (12). Thus, the presence of the suicide exon effectively eliminates gene expression, and its conditional splicing regulates expression of the encoded ORF. The coupling of alternative splicing to mRNA quality control pathways is conserved as a regulatory mechanism in diverse eukaryotic organisms, including plants, fungi and metazoans (13), so this method for transgene regula- tion could have broad applicability. So far, a few conditional splicing systems have been constructed that regulate gene expression in budding yeast (14) and mammalian cells (15,16). These studies were performed either on single reporter constructs or in the context of gene fusions which introduced extraneous sequences to the N-terminus of the ORF, similar to minigene reporters used in splicing assays (17). In addition, a natural riboswitch has been found that regu- lates gene expression through alternative splicing in response to thiamine pyrophosphate (TPP) in plants and ﬁlamentous fungi (18–21). The untranslated regions (UTRs) containing the TPP riboswitch have been appended to reporter constructs. However, the level of gene activation was modest even in thiamine-deﬁcient plant lines, as levels of the spliced product which gives higher gene expression was increased 7-fold upon thiamine depletion (20). Thus, it has remained unclear whether conditionally spliced transgenes can be reliably designed for robust gene activation, such that this method is generalizable to any gene of interest. Pre-mRNA splicing reactions can be sensitive to sequence context, as even single nucleotide polymorph- isms have been shown to cause aberrant splicing in some genes (22). Thus, maintaining the ﬁdelity and regulation of alternative splicing within diverse coding sequences could be quite challenging. In this study, we have found that a natural splicing cassette with a suicide exon could be