4706 Nucleic Acids Research, 2012, Vol. 40, No. 10 substitution at position 3 was not tested because it would generate a premature termination codon within the coding sequence. Mutations at the more highly conserved positions 2 and 1 lead to partial or full loss of splicing ﬁdelity and regulation. M3-M7 constructs harboring various muta- tions at position 2 were characterized as semi-functional. These mutants exhibit aberrant splicing but still some splicing to SP-I and SP-II, which leads to overall lower ﬂuorescence induction relative to WT. Sequence analysis of the observed aberrant spliced products, SP-III and SP-IV, showed that they are generated by improper usage of an upstream 50-ss or intron retention, respectively (Figure 2A). This is consistent with the idea that these mutations weaken recognition of the 50-ss. The M8 con- struct, which deliberately substitutes the least frequently observed nucleotides at positions 1 through 3, is fully non-functional. This mutant displays no induction of protein expression and only aberrant spliced products (SP-IV and SP-V), consistent with an impaired 50-ss. In contrast, alterations near the 30-ss do not appear to affect splicing activity, as evidenced by the M9-M12 con- structs. Even the M12 construct, in which positions +1 and +2 are substituted with the least frequently observed nucleotides, maintains full splicing activity. We did not rigorously test mutations to position +3 because no nu- cleotide conservation was observed at this position (28). In addition, M13-M17 constructs conﬁrm that double mutations to the bordering codons are tolerated as well as the corresponding single mutations (Figure 2D and Supplementary Figure S2). Taken together, these mutagenesis results suggest that full splicing activity for the splicing cassette requires a 50 context that extends to positions 1 and 2, but that there is no apparent requirement for the 30 context past the canonical 30-ss. In order to validate these surprisingly minimal exonic sequence requirements, we further analyzed the activity of the splicing cassette in ORF contexts corresponding to several of the tested mutations. The predicted functional contexts E/R, K/R and E/S were tested in the ORFs for fLUC or abscissic acid 80-hydroxylase (CYP707A3) as a representative plant gene. In good agreement with the results for WT, M1 and M9 constructs, the splicing cassette in these ORFs displayed full splicing activity and (analyzed by enzyme activity for fLUC only) increased protein expression in response to AtL5 (Figure 3A–C). The predicted non-functional context C/R was also tested in the ORF for phytoene synthase (PSY). Similar to the result for the M8 construct, the splicing cassette in this ORF is deregulated and gives related aberrant spliced products (Figure 3D). These data provide strong evidence that the mutagenesis results can reliably predict splicing activity for the P5SM cassette in other ORF contexts. If predicted functional contexts are limited to the 12 amino acid pairs that have been validated as giving full splicing activity (Figure 2 and Supplementary Figure S2), bioinformatics analysis suggests that the splicing cassette can be inserted within the ﬁrst half of 93% of annotated ORFs in the A. thaliana genome. However, our results also show that the 30-ss context appears not to inﬂuence cassette activity (Figure 2). The minimal exonic context for full mainten- ance of splicing ﬁdelity and regulation appears to be an AG immediately upstream of the 50-ss. An in-frame NAG codon would encode any of the amino acids Glu, Lys or Gln. If we consider all ORFs that contain one of these three amino acids within the ﬁrst half of their coding regions, the genome coverage estimate rises to 99.7%. Rational adaptation of a species-divergent P5SM element gives robust, orthogonal gene activation The natural P5SM cassette appears to maintain splicing ﬁdelity and regulation within diverse coding sequences. However, one potential drawback is that basal activation from endogenous L5 contributes to background ﬂuores- cence and leads to relatively modest ( 4–7-fold) levels of gene induction by AtL5 (Figure 1D and 3C). To eliminate basal activation, one might consider knocking out the en- dogenous protein, but since L5 is a component of the ribosomal machinery, this is expected to be highly detri- mental. Also, it is preferable to avoid requiring a speciﬁc genotype for use of the splicing cassette. Alternatively, we reasoned that the P5SM RNA element from a more divergent plant species might be less respon- sive to the endogenous L5 as compared to the A. thaliana P5SM RNA element. Both A. thaliana and N. benthamiana, the model species in which the transient ex- pression assays (see ‘Materials and Methods’ section) have been performed, are dicots. Thus, we replaced the P5SM RNA element in the reporter construct eGFP-P5SME/R with one derived from the monocot O. sativa, which was termed OsP5SM. We found that splicing activity is main- tained for the new construct eGFP-OsP5SME/R, which also exhibits a higher level of induction by OsL5 than AtL5 (Figure 4A). This construct was expected to give lower background ﬂuorescence due to reduced activation by the endogenous L5. Instead, higher background ﬂuor- escence is observed (Figure 4C, left side of leaves), corres- ponding to an increased ratio of SP-I to SP-II in the uninduced sample (Figure 4D). This unexpected result can be rationalized in light of the proposed regulatory mechanism for how the P5SM-L5 interaction inﬂuences alternative splicing (23). Previous data have suggested that L5 protein binding to the P5SM RNA element acts to displace an exon-deﬁning splice factor from the L2 loop, which results in exon skipping. The L2 loop of the A. thaliana P5SM RNA element has an extended purine-rich sequence that is shortened in the rice element (Supplementary Figure S3). It had been shown that substitution of this purine-rich sequence with the sequence UC leads to constitutive exon skipping (23). Thus, we hypothesized that swapping in the OsP5SM RNA element not only dis- rupted binding to endogenous L5 but also to the putative splice factor. Loss of exon deﬁnition due to a reduction in splice factor binding or another mechanism involving the L2 loop could explain the increase in SP-I and high back- ground ﬂuorescence. To address this possibility, we con- verted the sequence of the OsP5SM L2 loop to match the