branchpoint signal is sufﬁcient for proper splice site recog- nition. Taken together, it is possible that the P5SM splicing cassette will regulate the expression of any gene with the dinucleotide AG in its coding sequence, which effect- ively would mean that any gene can be regulated in a trace- less fashion. The splicing cassette was reliably engineered into three different ORFs, eGFP, fLUC and CYP707A3. The targeted ORFs did not originally possess introns at the site of cassette insertion, but in each case the suicide exon was spliced with no observable loss of ﬁdelity. All of the predicted functional contexts we have tested main- tain activation of alternative splicing by L5, demon- strating that the splicing cassette retains the necessary cis regulatory elements. A 2-fold difference in expression level was observed between the E/R and K/R contexts in the fLUC gene, regardless of the presence or absence of AtL5 (Figure 3C). Thus, there can be a slight sequence context effect that is independent of alternative splicing regulation, so we recommend testing two or three different contexts in the same ORF for optimal function. This strategy is practical because most coding sequences contain multiple candidate sites for cassette insertion, and overlap extension PCR employing different primers can be used to generate all of the desired constructs at the same time. Essentially no ﬂuorescence was measured for the eGFP- HyP5SM reporter in the absence of OsL5 induction (Figure 4B), even though the strong, constitutive CaMV 35S promoter was used to drive transcription and the ex- periment was performed in wild-type plants. This result shows that conditional splicing does not require the use of weak promoters or specialized genotypes. Furthermore, this result demonstrates that a plant-derived regulatory element could be employed in plants with almost no leaky expression. Basal activation by endogenous L5 was eliminated through rational improvements to the P5SM RNA element that were devised from our understanding of its role in alternative splicing. The splicing cassette har- boring the HyP5SM RNA element was selectively activated by OsL5 over AtL5 (Figure 4A). Restoration of the purine-rich L2 loop was required to promote default exon retention, which is consistent with the obser- vation that purine-rich motifs act as exonic splicing enhan- cers in A. thaliana (32). The ribosomal protein L5 for N. benthamiana has not been fully sequenced, so a direct assay has not been performed, but the little to no back- ground induction observed in wild-type N. benthamiana supports that NbL5 also does not recognize the HyP5SM RNA element. It is not immediately obvious which nucleotide changes to the RNA element are responsible for the discrimination in binding of OsL5 versus AtL5. There are very few dif- ferences in sequence between the original and hybrid P5SM in the regions that are homologous to 5S rRNA (the L2/P2 and P3c/L3 regions, Supplementary Figure S3). More differences are observed in the P3a/b stem, which in general exhibits higher sequence variability between representatives (23). Structural information about the P5SM-L5 complex will be needed to ascertain the molecular interactions which dictate recognition of the Nucleic Acids Research, 2012, Vol. 40, No. 10 4709 RNA element by the L5 protein from O. sativa but not from A. thaliana. Because L5 is not a general splice factor and instead interacts speciﬁcally with the P5SM element, the activa- tion of transgene expression by alternative splicing is highly robust and selective. Coexpression of OsL5 in- creases the levels of the reporter protein 97-fold (Figure 4B) but has no effect on the levels of another ﬂuorescent protein, DsRed2, used as a normaliza- tion standard in all other experiments (Supplementary Figure S4). This result is particularly impressive given that only a single copy of the P5SM RNA element is present in the splicing cassette. The induction of protein expression is comparable to that obtained using a condi- tional promoter harboring six copies of the GAL4 upstream activating sequence upon dexamethasone treat- ment (33). In conclusion, we have adapted a natural splicing cassette to serve as a portable regulatory element that robustly controls gene expression via alternative splicing. Gene regulation by the suicide exon is effectively traceless, as induced exon skipping affords the original ORF with at most one or two synonymous codon substitutions. This, along with the minimal requirements for sequence context, makes the regulation of any gene of interest quite facile using the P5SM splicing cassette. Thus, we have shown that conditional splicing can be a general and effective mechanism for transgene regulation. It is envisioned that the ability to combine DNA- and RNA-level regulation will enable novel strategies for controlling the expression of single genes with multiple promoters and for coordinating the expression of genes without the use of homologous promoters (Figure 5). We are currently Figure 5. Strategies for transgene regulation that combine conditional promoters and splicing cassettes. (A) Concept for promoter stacking with the P5SM splicing cassette. Expression of a single gene is regulated directly by its own promoter (P2) and indirectly by the promoter driving expression of the inducer (P1). Introduction of additional splicing cassettes would enable stacking of more than two promoters. (B) Concept for coordinated regulation of transgenes with the P5SM splicing cassette. A single conditional promoter (P1) drives ex- pression of the inducer, which can target multiple suicide exons. The DNA sequences of the splicing cassettes can be non-homologous, as the recognition element is an RNA structure, to avoid gene silencing. Different constitutive promoters (P2–P4) are used for the individual transgenes, as expression is instead coordinated by conditional alterna- tive splicing.