4704 Nucleic Acids Research, 2012, Vol. 40, No. 10 iSCRIPT reverse transcriptase (Bio-Rad) and oligo d(T) primers following the manufacturer’s instructions. Spliced products were ampliﬁed from cDNA samples by PCR with Taq polymerase (NEB) using target-speciﬁc primers (Supplementary Table S1). Spliced products were visualized by agarose gel electrophoresis and conﬁrmed by sequencing after gel puriﬁcation and TOPO cloning (see Supplementary Figure S6 for sequence maps of spliced products). RT–qPCR analysis was performed on the Bio-Rad CFX96 instrument using spliced product-speciﬁc primers (Supplementary Table S1) and SsoFast EvaGreen Supermix (Bio-Rad). The speciﬁcity of spliced product ampliﬁcation was conﬁrmed by melting curve analysis and visualization of PCR products on an agarose gel. Control samples in which no reverse transcriptase was added to the reaction also were analyzed after PCR to conﬁrm the absence of DNA contamination. Transcript abundances for different samples were calculated using standard curves determined for each target. Primer efﬁciencies were determined to be within the range of 100 ± 5%. Relative transcript amounts were normalized to the relative amount of DsRed2 transcript and the sample with the control LUC was set to 1. For biological replicates, no more than two leaf samples were taken from the same plant. Luciferase assay Fireﬂy luciferase (fLUC) enzyme activity was measured using the Luciferase Assay System (Promega) according to the manufacturer’s instructions. N. benthamiana leaf tissue samples were collected and analyzed 3 days post-inﬁltration. Tissue lysate samples were normalized to total protein content determined by using the detergent-compatible (DC) protein assay (Bio-Rad), and relative luminescence was measured in triplicate using a GloMax 96 Microplate Luminometer (Promega). Fold in- duction was determined relative to samples without coexpression of inducer. Bioinformatics The TAIR9 release of 33 201 annotated protein coding se- quences for A. thaliana was inputted into a MySQL database table. A database query was made for each pair of amino acids (corresponding to the bordering codons) within the ﬁrst half of protein coding sequences. Next, a database query was made counting the number of protein entries not containing any of the amino acid pairs within the ﬁrst half of the coding sequence. This number was subtracted from and divided by the total number of protein coding sequences to calculate the proteome coverage percentage. Similar queries were used to calculate the percentage of protein entries containing an E, K or Q amino acid within the ﬁrst half of the coding sequence. RESULTS A natural splicing cassette bearing a suicide exon can be employed to regulate other genes We and others have previously reported the discovery of the plant 5S rRNA mimic (P5SM), an RNA element residing within a highly conserved, alternatively spliced suicide exon that controls expression of transcription factor IIIA (TFIIIA) in land plants (23,26). We have shown that TFIIIA expression is activated by skipping of the suicide exon, which is induced by ribosomal protein L5 binding to the P5SM RNA element (23). Our initial design for a splicing cassette conservatively included not only the P5SM exon (175 nt in length) and ﬂanking intronic regions (150 and 98 nt in length) from the A. thaliana TFIIIA gene, but also the two bordering codons, which encode amino acids Glu (E) and Arg (R), respectively (Figure 1A). This design was intended to balance preserving the relative splice site strengths, in case they were important for alternative splicing activity, with minimizing the gene context requirements. Using the overlap extension PCR method (24), the above splicing cassette, called P5SME/R, was inserted in place of the codons for E-96 and R-97 within the coding sequence for enhanced green ﬂuorescent protein to generate the reporter construct eGFP-P5SME/R (Figure 1A). Thus, outside of the inserted cassette, the only change to the coding sequence of eGFP was a silent mutation of codon 97 from CGC to AGA (both encode Arg). This codon was changed in order to match the sequence surrounding the 30-splice site (ss) in TFIIIA. The function of the splicing cassette in this non-native gene context was compared to that for the control con- struct, TFIIIA-eGFP, in which the cassette remains in the native TFIIIA context with eGFP fused to the C-terminal end (Figure 1A). RT–PCR analysis of eGFP-P5SME/R detected only the expected exon-skipped (SP-I) and exon-retained (SP-II) spliced products (Figure 1B). The SP-I transcript was identical to the original eGFP coding sequence, except for the silent mutation, and its levels were increased 1.5-fold by coexpression of A. thaliana L5 (AtL5), similar to previous observations for TFIIIA-eGFP (Figure 1C) (23). The decrease in SP-II does not match the increase in SP-I, most likely because the abundance of the exon-retained spliced product is governed by NMD; this agrees with previous observations that the SP-II of TFIIIA is degraded (27). Consistent with the RT–PCR analysis, increases in protein ﬂuorescence with AtL5 induction were com- parable between eGFP-P5SME/R and TFIIIA-eGFP (Figure 1D). The representative leaf scans for each con- struct (Figure 1E) show similar AtL5 induction of eGFP ﬂuorescence, as well as some background ﬂuorescence induced by endogenous L5. These results support that the splicing cassette is fully functional within a foreign context—the eGFP coding sequence—with conservation of only the immediate bordering codons. We further inserted P5SME/R into several A. thaliana protein coding sequences, in place of E/R sequences nat- urally present within the ﬁrst half of each ORF. In each of these constructs, splicing ﬁdelity and regulation appear to be maintained, although some splicing intermediates were observed that correspond to unspliced and partially spliced pre-mRNA (Supplementary Figure S1). It is unclear whether these intermediates build up to detectable levels due to slower spliceosome processing or RNA deg- radation, but we later determined that they are not