Engineering a Bistable Splicing Switch
Splicing factors in metazoans commonly regulate their own levels by autogenous feedback loops. One example is the Drosophila Sxl protein, which is expressed in females but not in males to control numerous sexually dimorphic characteristics in the fly. Sxl protein promotes the splicing of the female form of its own mRNA by repressing use of an exon that is included in males. The male splicing pattern creates a premature stop codon that triggers the decay of the male isoform. Thus, when Sxl protein is expressed (as in females) Sxl mRNA is processed to produce more Sxl protein, whereas in males, no Sxl expression means no Sxl mRNA. This situation creates a female state (Sxl+) and a male state (Sxl–), which formally represent epigenetic states controlled at the level of RNA processing. We wish to model the requirements for establishing and the dynamics for maintaining the kind of bistable splicing switches that we see in metazoan systems using a simple manipulable cell.
To begin, we have used the yeast Saccharomyces cerevisiae and its MER1 protein, an RNA binding protein that activates splicing of four mRNAs during meiosis, through a splicing enhancer sequence found near the 5′ splice site of MER1-activated introns. MER1 itself is an intronless gene; however, we have created a MER1 gene that has a MER1-activated intron. Like Sxl in flies, the continuing expression of MER1 in these engineered yeast cells is dependent on MER1, and our hypothesis is that yeast carrying this gene may exist in one of two states: a MER1+ state, in which MER1 expression is sufficient to promote continued splicing of MER1 pre-mRNA expression of MER1 and maintenance of the MER1 state, and a MER1– state, in which insufficient MER1 is present to promote MER1 splicing and expression. There is a low level of splicing that is independent of MER1 expression, thus we expect that stochastic fluctuations in MER1 transcription or other steps in MER1 gene expression might allow transition between the MER1+ and MER1– states.
This project will involve developing real-time markers of the MER1 state using fluorescent reporters containing MER1-dependent introns and measuring the relationship between MER1 expression and the stability of the MER1 states. The results will provide a framework for understanding the function and dynamics of bistable splicing switches and their role in maintaining epigenetic states of differentiation.