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."
Besides studying splicing mechanisms, Black is determining how signals from outside a cell regulate splicing. He discovered, for example, that the entry of calcium ions into neurons can alter the splicing of many pre-mRNAs, including those encoding the channels that allow calcium to enter the cell to begin with. Thus, calcium ions, by regulating alternative splicing, alter their own signaling properties. Black identified several short pre-mRNA sequences that respond to these calcium signals, and he is working to identify the corresponding RNA-binding proteins. This process is particularly interesting in mature neurons, where calcium signaling plays a major role in controlling synaptic activity.
His group is also investigating the effects of alternative splicing on developing neurons. They discovered that the progenitor cells that give rise to neurons express an RNA-binding protein called PTB. As those cells differentiate into neurons, the level of PTB drops as it is replaced with a related protein called neuronal PTB (nPTB). Black showed that the switch from one RNA-binding protein to the other reprograms splicing on a grand scale during neuronal differentiation. In a group of important mRNAs, the splicing of a specific set of exons is changed. The modified proteins resulting from this switch are involved in cell adhesion, the assembly of the cytoskeleton, and other important developmental processes.
This work also had a surprising twist: the production of one PTB protein is regulated by the other, because the presence of PTB in the neuronal progenitor cell prevents expression of nPTB. The loss of PTB during differentiation is sufficient to induce nPTB expression in the neuron. "The nPTB message is there, but its splicing is changed, and its translation is suppressed by the presence of PTB," Black says. "So there's a lot of interplay among these different RNA-binding proteins in regulating each other."
In this work and increasingly in the future, a more global view of splicing regulation is becoming possible. "We know that particular proteins or stimuli will change a whole bunch of exons," Black says, "but it's not so clear how these ensembles of splicing events are important to a particular cellular function. We and others in the field are hoping to learn more about that in the nervous system before long."
The goal of this work is primarily to understand how alternative splicing is accomplished and how it affects cells. But errors in splicing are implicated in many neurological and other inherited diseases. "To understand how these mutations cause deleterious effects, we first need to understand how cells recognize exons and assemble spliceosomes," Black says.
In very recent work, Black's group has been developing methods for identifying small molecules and drug candidates that alter the splicing of particular disease exons. "This has been a new way of thinking for me," Black says. "I started with very mechanistic interests, but have grown in a more biological direction with time."