The goal of this pilot proposal is to develop a system in which the expression of a reporter gene can be conditionally and reversibly inactivated in mammalian cells by regulating alternative splicing. This will be achieved by controlling the activity of an engineered splicing regulatory protein. Members of the SR protein family can function as splicing regulators by binding to alternative exons and enhancing the utilization of upstream splice sites. SR proteins are modular, containing separable RNA binding and splicing activation domains. In fact, the splicing activation domain can retain its function when fused to a heterologous RNA binding protein. Moreover, the splicing activation domain can even function when contained on a separate polypeptide than the RNA binding domain, provided that the two proteins can be physically juxtaposed. This has been achieved by fusing the RNA binding and splicing activation domains to the proteins FKBP and FRB, respectively. FKBP and FRB are two human proteins that do not interact with one another, but can be linked together by the small drug rapamycin (rap), which can simultaneously bind to both proteins. Rap can therefore be used to control the association of the RNA binding domain with the splicing activation domain, and thus regulate the alternative splicing of exons that contain binding sites for the regulator. Here, we propose to use this system to control alternative splicing in tissue culture cells. Specifically we will develop reporter genes in which a constitutively spliced intron disrupts the open reading frame for a reporter gene. Within this intron, we will insert an alternative exon containing in-frame stop codons and binding sites for the splicing regulator. Thus, when the alternative exon is included, no functional reporter will be produced. Experiments are described in each component of the alternative exon is optimized independently. Expression vectors encoding the RNA binding domain-FKBP and splicing activation domain-FRB fusion proteins will be used to test the ability of rap to inactivated expression of the reporter genes. Finally, the induction and decay kinetics of rap-induced alternative splicing will be determined. These experiments will result in the development of a novel system for conditionally and reversibly controlling gene expression. If successful, this system will be extended to conditionally and reversibly inactivate gene expression in transgenic mice. The availability of this method to study genes involved in bone development would significantly enhance our understanding of osteogenesis and will allow experiments to be performed that are not technically possible with the methods available today.
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