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Understanding the Mechanics of Splicing

Douglas Black in his UCLA lab. | Photo by Rebecca Fairley Rainey

Take a tour of the sprawling rooms and spaces of Douglas Black's lab, and you'll get a crash course on an astonishing array of equipment. On one side of the hallway there are benches for nearly a dozen researchers and students, along with an FPLC, a machine that purifies proteins. Next door, there's equipment to sort molecules, as well as an incubator with roller bottles. Across the hallway, tucked into its own room, near a group of centrifuges, there's a high-density device to sequence DNA. Through another doorway is a mouse dissection area, along with stacks of mouse cages.

This is where AAAS Fellow Douglas Black studies a process called RNA splicing. It's where Black Lab UCLA researchers examine the molecular mechanisms of this process and the role it plays in biology and disease.

"Across all cell types and tissues," Black said, "the splicing process has turned out to be very important." The "misregulation" of splicing, he said, "is now implicated in many human disorders from cancer to neurodegeneration." 

His research into the general mechanism of splicing has provided insights into rare neurological diseases such as Duchenne muscular dystrophy and spinal muscular atrophy, and possibly some forms of autism and epilepsy.

"Until we understand splicing," Black said, "we can't really interpret a genome and make use of the information it provides."

"Besides broad disorders of splicing regulation," Black explained, "a large number of individual human disease mutations alter the sites at which splicing occurs in an RNA, leading to a failure to splice correctly in a single gene."

In his work over the last 25 years, Black has developed methods to study splicing and identified cellular components that regulate it. He has learned how certain RNA-binding proteins that regulate splicing are required for the maturation of neurons. Other splicing proteins control how mature neurons respond to signals from other cells.

Along with Manuel Ares, a professor of molecular, cellular and developmental biology at the University of California at Santa Cruz, Black developed the first microarrays - a standard tool for testing DNA - that allowed simultaneous measurement of multiple changes in splicing.

RNA splicing allows each gene to encode multiple proteins and gives cells an additional mechanism for regulating protein production. To understand what Black described as "the programs of splicing regulation," his lab created several lines of mice; each one with a specific mutation in one of its splicing regulators.

This work, Black said, demonstrated that "PTB" proteins had "profound effects on early neuronal development." Mutations in a protein called "PTBP2" caused a failure of neurons to mature. Those mice died at birth or shortly after weaning, depending on how much of the brain was affected.

In contrast, mutations in another group of proteins, called "RBFox" proteins, "altered the function of mature neurons and their ability to signal to other neurons," Black wrote. "In the case of loss of the RBFox1 protein, cells become hyperexcitable and mice with this mutation are prone to seizures.

"These studies," he said, "along with those from other groups, demonstrated that RNA binding proteins are playing multiple key roles in neuronal development and function."

Beyond this work, the Black Lab seeks to understand how RNA binding proteins do their jobs. Using biochemical methods, it has "identified molecules that affect the process, and determined their interactions with other parts of the cell."  

Modern genomic methods have led to an "explosion of information" on regulatory splicing proteins, and the identification of splice-site mutations in human disease is "increasing dramatically with the sequencing of patient genomes."

These are potentially lifesaving lessons that might one day help patients whose diseases have not yet been explained by genetic science.