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The Spliceosome, Alternative Splicing, and Genomics
The work of the Ares laboratory centers on the mechanisms and regulation of splicing. Splicing is required to remove intron sequences from pre-mRNA and create coding sequences for translation. Yeast has been the laboratory's organism of choice for these studies because it offers simple, powerful, genetic approaches and has a splicing machinery similar to that in mammalian cells. In addition, the yeast genome is completely sequenced, the location of nearly every intron is known, and genes for most splicing factors have been identified. This provides unique advantages for the study of splicing. Measuring Splicing Everywhere Ares' laboratory exploits this knowledge by building microarrays that measure splicing of every yeast intron in parallel, and determing the effect of mutations or perturbations in the environment on splicing. They have recently created a compendium of microarray data using these arrays and more than thirty different deletions of genes involved in mRNA metabolism. As the human genome is completed, they are turning their attention to genome-wide studies of splicing in humans. It is estimated that more than 50% of human genes produce alternatively spliced mRNAs. These alternative mRNAs can code for distinct proteins with alternative functions, meaning that the regulation of alternative splicing is necessary to correctly interpret the genome sequence. Expression profiling methods that resolve these mRNA forms will be necessary to understand genetic processes such as cancer. Ares and his colleagues have already built and are testing a small array designed to measure changes in alternative splicing of RNA from tumor suppressor genes and oncogenes. Biochemistry of Splicing Regulation The Ares laboratory also studies how small nuclear RNAs function with snRNP proteins during splicing using a combination of genetics and biochemistry. Because of the high conservation of snRNA and snRNP proteins between yeast and mammals, results from yeast have immediate ramifications for the understanding of splicing in humans. Ares' goal is to analyze the basic events during the early steps in splicing in order to understand how they are regulated. For example, the positive splicing regulator Mer1p activates splicing of introns in three other genes during meiosis. Mer1p binds a splicing enhancer located in each of these introns and also binds to the U1 snRNP. The laboratory is currently attempting to determine how Mer1p accelerates splicing of these introns. In a second project, they are determining the mechanism by which the ATP-dependent step of U2 snRNP binding to the pre-mRNA is catalyzed by the ATP-dependent RNA helicase Prp5p. The ATP-dependence of Prp5 function is regulated by Cus2p and U2 snRNA, and is a key step in the spliceosome assembly pathway. Drug Targets Everything is a drug target - and splicing proteins are no exception!
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