An Alternative Splicing Platform for Programming Protein Function

Our ability to effectively engineer biological systems is limited by the tools and strategies available to detect, transmit, and control molecular information. While a number of RNA-based tools have been developed for controlling gene expression, molecular platforms that support more sophisticated mechanisms of control, such as reprogramming protein form and function, are needed to advance the scale and complexity with which we can construct and interact with biological systems. Alternative splicing is a naturally occurring process in eukaryotes that generates multiple protein isoforms from a single coding region by altering the ways in which exons are joined together. Alternative splicing is a major source of protein diversity and greatly increases genomic coding capacity, such that over 95% of multiexon genes in humans are alternatively spliced. A platform for programming alternative splicing has the potential to broaden current regulatory capabilities by providing a strategy to dynamically program protein function. We have developed a design framework that supports the modular and extensible implementation of an alternative splicing regulatory platform. We have constructed and characterized an intron framework that produces defined mutually exclusive alternative splicing patterns when placed in the context of various exon sequences. By integrating ligand-responsive synthetic RNA switches into this intron framework we demonstrate the conditional regulation of alternative splicing patterns. This alternative splicing platform has been used to conditionally link functional domains in modular proteins, such as TALE-TFs, dynamically program protein function, and ultimately engineer complex regulatory networks in higher organisms.