Paul Boutz' Page

A NEURAL-SPECIFIC SPLICING DEPENDENT GFP REPORTER

Paul L. Boutz and Douglas L. Black
Dept. of Microbiology, Immunology, and Molecular Genetics and Howard Hughes Medical Institute, UCLA, Los Angeles, CA 90095

Inclusion of the N1 exon of the c-src transcript is specific to neuronal cells and depends on multiple regulatory elements. Biochemical experiments have identified protein factors that influence the splicing of the exon, including hnRNPs F and H, PTB, and KSRP. However, the mechanism by which these sequence elements and proteins confer cell-type specificity to the N1 exon is not well understood. An in vivo assay for identifying factors controlling N1 splicing would be useful in both confirming the involvement of suspected splicing regulators and for identifying new factors. We have previously shown that a src minigene construct containing the N1 exon and flanking intron and exon sequences exhibits neural-specific splicing regulation both in vivo and in vitro. Based on this substrate, we have designed a reporter construct that can be used to screen for activators of N1 exon inclusion in vivo. The reporter gene, green fluorescence protein (GFP), is situated within the src minigene in such a way that it is translated out of frame when N1 is spliced out, as normally occurs in non-neuronal cells; in contrast, the inclusion of N1 shifts the reading frame, resulting in the production of functional GFP. Thus cells which follow the neuron-specific splicing pathway can be identified by their GFP fluorescence and isolated. We have tested the reporter construct by transient transfection in several cell lines and found it to be highly regulated, producing functional GFP only in neuronal cell lines. We are currently characterizing stable cell lines which express the reporter transcript. We plan to use this assay to screen a neuronal cDNA library for cDNAs that activate the neuron specific splicing pathway in non-neuronal cells. We can also employ this reporter to investigate the nature of the intronic splicing enhancer downstream of the N1 exon. By replacing the enhancer within the src GFP minigene with a stretch of randomized sequence, we can use expression of the GFP reporter to identify sequences that influence the N1 splicing pattern. This approach should give us new insight into what constitutes an intronic enhancer, what proteins interact with it, and whether its components are sequence specific, structural, or both.