Published online 6 February 2012 Nucleic Acids Research, 2012, Vol. 40, No. 10 4701–4710 doi:10.1093/nar/gks032 Transgene regulation in plants by alternative splicing of a suicide exon Scott F. Hickey1, Malathy Sridhar1, Alexander J. Westermann 2 , Qian Qin1, Pooja Vijayendra3, Geoffrey Liou3 and Ming C. Hammond1,3,* 1Department of Chemistry, University of California, Berkeley, CA 94720, USA, 2Institute for Molecular Infection Biology, University of Wu¨ rzburg, D-97080 W u¨rzburg, Germany and 3Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA Received November 23, 2011; Revised January 4, 2012; Accepted January 6, 2012 ABSTRACT Compared to transcriptional activation, other mech- anisms of gene regulation have not been widely ex- ploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surpris- ingly minimal sequence requirements for the main- tenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation. INTRODUCTION Alternative splicing of precursor mRNAs (pre-mRNAs) is an important and widely conserved mechanism for increasing protein diversity and for gene regulation. In plants and other eukaryotes, tissue-speciﬁc and development-speciﬁc expression of genes is regulated by alternative splicing (1–3). Changes in pre-mRNA splicing of plant transcripts have been observed in response to stress conditions, including growth in cold temperature or under drought conditions (4). Alternative splicing also has been shown to contribute to diverse physiological processes in plants, including regulation of circadian rhythm and the defense response to pathogens (5,6). Altogether, it has been estimated that between 20% and 30% of expressed genes are alternatively spliced in Arabidopsis thaliana and Oryza sativa (rice) (7,8). While alternative splicing appears to be extensively employed in natural regulatory systems, it has not been generally applied to the conditional expression of trans- genes. Currently, transgene regulation is based almost ex- clusively on transcriptional activation. This is due, in no small part, to the ease of placing a promoter sequence upstream of any gene of interest, which requires little to no characterization of promoter elements and no alter- ation of the coding sequence. However, many conditional promoters suffer from issues such as leaky basal expres- sion, pleiotropic effects and species speciﬁcity (9). Promoters are also difﬁcult to combine serially in order to generate complex regulatory patterns, as cross-talk between different promoter elements often leads to unpre- dictable effects on gene expression (10). Furthermore, use of multiple copies of identical promoters to coordinate regulation of several genes can trigger gene silencing (11). Thus, one motivation for developing a method for transgene regulation based on alternative splicing is that problems with existing conditional promoters may be ameliorated by combining DNA- and RNA-level regula- tion. Another advantage of a conditional splicing system is that the gene can be regulated and still remain under the control of its endogenous promoter. There may be add- itional beneﬁts to cotranscriptional mechanisms of regu- lation which have yet to be explored because a general conditional splicing system has not been developed. An important advantage to the use of conditional pro- moters is that it does not change the sequence of the translated open reading frame (ORF). However, alterna- tive splicing of a cassette harboring a suicide exon can also be considered to operate in a traceless manner (Figure 1A). Exon skipping would generate a productively translated spliced product (SP-I) with the suicide exon cleanly excised from the sequence of the ORF. Alternatively, exon inclusion would introduce a premature termination codon that targets the spliced product (SP-II) *To whom correspondence should be addressed. Tel: +1 510 642 0509; Fax: +1 510 550 2879; Email: mingch@berkeley.edu The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.