Alternative splicing is an important cellular process that is highly regulated and impacts many fundamental cellular decisions and we only have a rudimentary understanding of how it is regulated, given the range of competing factors. The binding of RNA binding proteins (RBPs) to specific sites in pre-mRNAs plays a central role in regulating alternative splicing. Changes in the concentrations of RBPs that regulate splicing have been implicated in many human diseases including cancer, heart and neurological disease. To understand how alternative splicing is regulated over a concentration gradient, novel cellular dosing systems have been created with the splicing factor MBNL1. The MBNL1 dosing systems can be used to study alternative splicing up to a 20-fold range of protein. Next generation sequencing and global RNA structure probing approaches are being used to characterize how changes in MBNL1 concentration control alternative splicing. The biological implications of these studies are many and significant. The results from the MBNL1 dosing studies will provide a framework to understand and potentially predict splicing events that will change at low MBNL concentrations and do so quickly (steep slope) in different tissues during development. The dosing system can also be used to predict and model the behavior of splicing events in which the concentration of MBNL1 is altered and will help guide the selection of biomarkers for myotonic dystrophy.
Many trans and cis acting factors that control alternative splicing have been identified. The explosion of next-generation sequencing approaches have identified thousands of regulated splicing events (RNA-seq), and binding sites of many proteins which regulate alternative splicing have been identified via CLIPseq. An important question remaining for the field is: What are the rules governing the behavior of alternative splicing decisions? For example, what are the RNA elements (sequence and structure) that determine if alternatively regulated exons will respond at low concentrations or at high concentrations to a splicing factor, and will the splicing responses exhibit cooperative behavior or not? Addressing these questions is important for providing a framework for understanding how changes in splicing factor concentration can lead to disease. To address these questions, we have created cellular models that allow us to precisely titrate the level of an alternative splicing regulator, the Muscleblind-like 1 (MBNL1) protein. MBNL1 has been shown to regulate thousands of alternative splicing events and is important for the development of skeletal muscle, heart and the central nervous system. This regulation is highlighted by the primary role that MBNL proteins play in the disease myotonic dystrophy (DM), in which MBNL1 and its paralogs (MBNL2 and MBNL3) are sequestered by expanded CUG or CCUG repeat RNAs, resulting in aberrant RNA processing. The mis-splicing of MBNL targets has been shown to be responsible for causing some of the symptoms associated with DM, including the hallmark symptom myotonia. .
|Hale, Melissa A; Richardson, Jared I; Day, Ryan C et al. (2018) An engineered RNA binding protein with improved splicing regulation. Nucleic Acids Res 46:3152-3168|