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Research Summary One surprising result of the recent sequencing of the human genome was that the number of predicted genes is significantly lower than had been anticipated. Alternative pre-mRNA splicing is a powerful, important, and prevalent strategy that higher eukaryotes have developed to increase the number of different proteins encoded by their genome. Moreover, alternative splicing plays a major role in gene regulation, is frequently developmentally or tissue-specifically regulated, and can generate distinct protein isoforms with different functions. Even today, the molecular mechanisms governing alternative splicing are poorly understood, but the key regulatory proteins, as well as the mechanisms identified thus far, are well conserved between metazoan species. Hence, the use of the fruit fly Drosophila melanogaster, with its small and well-annotated genome, continues to be a powerful system with which to decipher the mechanisms involved in alternative pre-mRNA splicing. My laboratory is interested in understanding detailed mechanisms controlling alternative splicing. Future efforts concentrate on three major areas: 1) identification of genes and splicing events regulated by specific splicing factors, 2) identification of the protein factors and RNA sequence elements controlling specific alternative splicing events, and 3) characterization of the detailed molecular mechanisms controlling specific alternative splicing events. Global Characterization of the Alternative pre-mRNA Splicing Network One factor, multiple pre-mRNA targets My lab has developed a microarray platform aimed specifically at measuring global differences in the alternative splice junction used. This genome-wide strategy is used to identify genes whose alternatively spliced junctions are specifically regulated by individual splicing factors. The strategy that we have successfully developed is to knock-down a specific RNA binding protein using RNA interference (RNAi) in Drosophila tissue culture cells, purify RNA, and prepare cDNA that is hybridized to splice junction microarrays. This platform will enable us to identify specific genes regulated by individual members within different families and classes of splicing factors. Analysis of the affected genes will lay the foundation to identify the regulatory elements specific for each factor and will facilitate dissecting their mechanisms of action at the molecular level. One pre-mRNA target, multiple regulators A second important effort is the identification of the other factors regulating specific splice junction use. Our strategy is to create in vivo reporter systems where a specific alternative splice junction controls the expression of a reporter gene, such as EGFP or E. coli ß-galactosidase. Following transcription of the hybrid construct, splicing of the transcripts produce one splice variant in frame with the reporter and leads to expression of the reporter protein, while the other splice variant is out of frame and does not generate any protein product. This reporter system is then subjected to a high-throughput genome-wide RNAi screen and, in addition to identifying RNA binding proteins directly involved in the splicing reaction, this screen has the potential to identify all the components in a regulatory cascade, including factors such as protein kinases that might control the activity of the splicing factors, transcription factors required for the expression of the proteins controlling the alternative splicing junction, as well as signaling factors responsible for controlling the expression of the specific mRNA isoforms. Elucidation of the Molecular Details Controlling Alternative pre-mRNA Splicing Biochemistry of the regulation of alternative pre-mRNA splicing The splice junction microarrays provide us with numerous specific alternative splice junctions affected by individual splicing factors and will provide a means to engineer in vitro transcribed and radiolabeled RNA substrates that could be used for in vitro splicing and RNA binding assays in Drosophila nuclear extracts. Using the transcripts and junctions affected by individual members of the SR protein family, the cis-acting RNA elements will be identified through dissection of the pre-mRNA. Together with bioinformatic analysis looking at over-represented sequence motifs surrounding the splice sites regulated by individual proteins, these analyses should enable my laboratory to identify the specific binding elements recognized by individual factor. Protein composition of individual RNP complexes The protein composition of individual RNP particles assembled on specific pre-mRNAs can be determined by tagging individual pre-mRNA for affinity purification using the MS2 coat protein binding site and an RNA aptamer that binds specifically to the antibiotic tobromycin. The doubly-tagged RNA is expressed in Drosophila cell culture and affinity-purified, and the composition of the endogenous RNP complexes formed on individual pre-mRNA will be identified through tandem mass spectrometry. This will lead to a better understanding of the pathways and components involved in forming individual ribonucleoprotein complexes and what factors associate with specific transcripts to modulate alternative pre-mRNA splicing. Academic Appointment: Assistant Professor, Department of Pathology & Laboratory Medicine, The University of Kansas School of Medicine Selected publications
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Blanchette
Lab