Programmable mutually exclusive alternative splicing for generating RNA and protein diversity

Eric M Sawyer,Shireen S Rudina,Melina Mathur,Christina D Smolke,Sarah A Munro,Cameron M Kim

doi:10.1038/s41467-019-10403-w

Abstract

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

Highlights

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity
We developed a mutually exclusive alternative splicing (MEAS) intron framework comprising three introns interspersed between four exon positions, of which the second and third exons are mutually exclusive (Fig. 1a)
Alternative splicing-based approaches provide a powerful strategy for expanding coding capacity and for producing diverse RNA and protein isoforms from a single genetic device

Summary

Introduction

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms We apply this framework to program the DNAbinding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can be applied to program modular proteins and increase coding capacity of synthetic biological systems. Our work demonstrates a programmable and extensible MEAS platform for generating functionally diverse RNA and protein isoforms

Methods

Results

Conclusion