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Dr. Black is a Professor of Microbiology, Immunology, and Molecular Genetics at the University of California, Los Angeles and at the David Geffen School of Medicine at UCLA.
Dr Black's lab is interested in the regulation of pre-mRNA splicing and the biochemical mechanisms that control changes in splice sites. The sequences of metazoan genomes with their relatively low gene numbers have highlighted the question of how protein number can be expanded beyond the gene number for a complex organism.
Alternative splicing, in allowing the production of multiple mRNAs and hence multiple proteins from a single gene, is a major contributor to protein diversity. However, in spite of its key role in gene expression, this process is poorly understood mechanistically.
Alternative splicing is particularly common in genes expressed in the mammalian nervous system, where many proteins important for neuronal differentiation and function are made in diverse isoforms through controlled changes in splicing. Our lab works on a range of projects related to the control of pre-mRNA splicing in neurons. We aim to determine the mechanisms of action of splicing regulators and to understand their roles in neural development. We are focused on four regulatory factors: Polypyrimidine Tract Binding Protein (PTB), neuronal PTB, Fox-1, and Fox-2. PTB and nPTB are primarily splicing repressors, while Fox-1 and Fox-2 act to enhance splicing. Each of these proteins alters the splicing of a specific set of exons within the genome. In mechanistic studies, we examine the RNA binding properties of these proteins and analyze how they can alter spliceosome assembly. A second effort uses cell culture models and conditional knockout mice to understand how these proteins affect neuronal development. A final area of interest is in understanding how cell signaling pathways impact the splicing reaction. This project focuses on the effect of cell excitation on the splicing of ion channel transcripts and the role of this splicing in neuronal plasticity.
At one time, scientists thought that a gene would produce a single messenger RNA (mRNA) that would be translated into a single protein. But they now know that most genes have at least several different products and that one gene in the fruit fly, Drosophila, can produce as many as 38,000 proteins. The process that creates this situation is called alternative splicing, which allows cells to choose which parts of the long primary RNA transcript of a gene to include in the final mRNA. Through alternative splicing, different segments of RNA can be spliced together to produce mRNAs encoding different, but related, proteins.
Black says that alternative splicing is like having each sentence of a novel distributed through an encyclopedia-sized text of gibberish. In that case, you would need a method for recognizing the meaningful sentences and linking them together to read the novel. If certain important sentences were sometimes skipped and sometimes included, the meaning of the novel could be quite different. Similarly, the proteins resulting from alternative splicing often have different functions, and need to be made in specialized cell types, such as neurons or muscle. To find out how cells recognize the meaningful portions of the RNA and make choices about which segments to include in the mRNA, Douglas Black studies the ins and outs of splicing.
The splicing machine, an RNA-protein complex called the spliceosome, processes the precursors to mRNAs (pre-mRNAs) as they roll off the DNA of genes. These pre-mRNA molecules are exact copies of the transcribed portions of genes and contain regions (exons) that must be included in mRNAs. Exons, which generally code for a segment of protein, are separated by regions called introns, which must be removed from the mature transcript. The spliceosome discards the introns and splices together the exons to make mRNA.
During his graduate studies with HHMI investigator Joan Steitz at Yale University, Black became fascinated with RNA processing and how the spliceosome worked. "For me, the most interesting steps in the use of genetic information occur at the level of RNA," he explains.
During that time, it was becoming clear that alternative splicing could produce multiple mRNAs and hence multiple proteins from the same gene and that this process strongly affected the tissue-specific expression of specialized proteins. In several cases in Drosophila, it appeared that special pre-mRNA-binding proteins could determine where the spliceosome would cut and paste. "Proteins that bind to pre-mRNA alter the assembly of the spliceosomes so that they can either induce splicing at sites that are not normally recognized or prevent it at sites that would be used if those proteins were not there," Black says.
Black did his postdoctoral work with David Baltimore at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, developing methods for studying alternative splicing in the mammalian nervous system. "I have been extraordinarily lucky in my mentors, both for the training environments they created and their willingness to let me try new things," he says. "I advise my own students to look for those special places where everyone around you is full of ideas and diverse expertise. While your focus may be on RNA-binding proteins, you will ultimately use what you learn from your bench mates about leukemia, transcription factors, or autoimmune disease."
Black continued to study alternative splicing when he set up his lab at the University of California, Los Angeles in 1992. Early in his career, he devised a way to study alternative splicing in the test tube, making it possible to see how various protein regulators that bind to pre-mRNA affect splicing choices. Early models for splicing regulation had invoked a single splicing regulator that would shift the use of a splicing pattern away from a "default" choice. For one neuron-specific exon, however, Black found protein factors that inhibited the neuronal pattern of splicing as well as factors that stimulated it. This combination of positive and negative regulation has proved to be a common feature for tissue-specific splicing patterns. "It turned out that more components than we expected are involved," Black says. "Each exon that is spliced in or spliced out seems to be acted on by dozens of different factors."