4708 Nucleic Acids Research, 2012, Vol. 40, No. 10 Figure 4. Rational design of the P5SM RNA element leads to generation of an orthogonal splicing cassette. (A) Fold induction of protein expression quantiﬁed by eGFP ﬂuorescence for reporters harboring the OsP5SM or HyP5SM RNA elements within the splicing cassette. Induction with AtL5 or OsL5 was each measured in comparison to induction with LUC as a control. (B) Fluorescence of eGFP-HyP5SM without coinﬁltration with DsRed2 and with subtraction of background autoﬂuorescence for individual leaf samples. Fold induction by OsL5 relative to induction with LUC as a control is shown. Background subtraction gives a negative ﬂuorescence value (asterisk) for leaf 4 in the absence of OsL5 induction, so the fold induction could not be determined for this sample. (C) Representative whole leaf scans of eGFP ﬂuorescence for reporters harboring OsP5SM or HyP5SM. The right leaf halves coexpressed AtL5 or OsL5 and the left halves coexpressed LUC as a control. All samples except for the one labeled ‘No DsRed2’ also coexpressed DsRed2 on both leaf halves. (D) RT–PCR detection of spliced products for eGFP-OsP5SM and eGFP-HyP5SM upon induction with AtL5, OsL5, or LUC as a control. junction by some distance. In N. benthamiana, introduc- tion of the well-characterized Ls intron within the 30-UTR of an otherwise stable mRNA triggers NMD when the stop codon is 99-nt but not 28-nt upstream of the location of intron placement (12). This observation is con- sistent with the general rule established in mammals, which is that NMD is usually triggered if a stop codon is 50–55 nt upstream of an exon junction (29). The pre- mature termination codon in the P5SM suicide exon is 121 nt upstream of the exon junction, and the exon-retained spliced product of A. thaliana TFIIIA is subject to NMD (27). Regardless of gene context, the splicing cassette maintains the suicide exon at the same distance upstream of the exon junction, so its location probably does not need to be within the ﬁrst half of the ORF to trigger NMD. However, cassette insertion at a roughly central location will help ensure that each ORF section is of sufﬁcient length to be recognized as an exon. Second, we only consider in-frame cassette insertion sites which allow us to generate proper splice site contexts via synonymous codon substitutions. It is very likely that the conserved AG sequence upstream of the 50-ss does not have to be in-frame with the ORF, although this remains to be tested. Our observation that mutations in the extended 30-ss context have no effect is consistent with the spliceosome cycle as elucidated in mammalian systems, in which the 50-ss and branchpoint are ﬁrst recognized (30). The branchpoint region of the 30-intron can be readily identiﬁed, as it is 27-nt upstream of the 30-ss and conforms to the plant consensus, CURAY (31). Thus, it appears that mainten- ance of the distance between the 30-ss and the strong