Alternative splicing is a process by which multiple protein isoforms are generated from a single coding region by altering the ways in which exons are joined together during the splicing process. This pathway is used by cellular systems to both increase proteomic diversity from a limited number of genes and to precisely control gene expression. The ability to program splicing patterns will provide a powerful tool for researchers to probe and manipulate cellular function. However, the ability to engineer alternative splicing is limited by our lack of understanding of the cis-acting regulatory elements that control the levels of spliced isoforms. To begin to address this, we have developed and employed a novel in vivo Screening PLatform for Intronic Control Elements (SPLICE). We identified 125 unique ISRE sequences from a random 15 nucleotide library within mammalian cells using SPLICE. Bioinformatic analyses of the selected sequences reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors. The identified motifs are enriched in the introns of naturally-occurring spliced genes, supporting their biological relevance. In vivo studies demonstrate that the selected ISREs can exhibit combinatorial regulatory activity and that diverse splicing factors can be involved in the regulatory effect of a single ISRE. The flexibility of our screen has allowed for the selection of diverse intronic regulator sequences that vary in splicing efficiency, suggesting the possible use of these sequences to fine tune specific splicing events in order to achieve a desired level of gene expression. Encouragingly, the synthetic ISREs retain function when tested in a second cell type demonstrating their portability. Interestingly, our elements did not retain function in a second transcript, suggesting that their regulatory function is likely context dependent. The selected synthetic ISREs can be implemented for the precise regulation of splicing patterns of heterologous transcripts and SPLICE may be adapted to generate diverse groups of cell- and tissue-specific regulators of gene expression. The ISREs selected by SPLICE also offer a reduced library to explore the ability of chemical libraries to selectively target specific splicing events.

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