Environmental Factor, November 2009, National Institute of Environmental Health Sciences
Artificial Protein Can Direct Alternative Splicing of Genes
By Robin Mackar
What started out as a very basic research project looking at a fly protein, has now morphed into a plausible method that may help researchers better study and manipulate disease-associated gene activity.
Researchers at NIEHS have teamed with the Department of Pharmacology at University of North Carolina at Chapel Hill (UNC-CH) to develop artificial protein factors that can direct the alternative splicing of genes. Published online by Nature Methods, their paper (http://www.nature.com/nmeth/journal/v6/n11/abs/nmeth.1379.html) provides scientists with a new strategy for studying factors that regulate splicing or other RNA processing pathways. It also demonstrates the ability of one such factor to make cancer cells more susceptible to chemotherapy through manipulating alternative splicing of cancer-related genes.
Alternative splicing is an important and common occurrence that enables genes in humans and other organisms to make the variety of proteins needed to carry out the functions of cells throughout the body. More than 90 percent of human genes undergo alternative splicing, and the disruptions of splicing have been shown to cause human disease.
To generate the RNA-binding module, the researchers used a human version of a Drosophila fruit fly protein, part of a family of proteins called PUF proteins. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants. "We really didn't know much about the human protein at the time when we first began to study this family of proteins," said Traci M. T. Hall, Ph.D., principal investigator in the Macromolecular Structure Group at NIEHS (http://www.niehs.nih.gov/research/atniehs/labs/lsb/ms/index.cfm) and co-author on the paper with lead author Zefeng Wang, Ph.D., assistant professor at UNC (https://www.med.unc.edu/pharm/people/primaryfaculty/zefeng-wang). "I used to joke that we were using the human protein as a model system for the fly."
The NIEHS and UNC groups have used the technology to create unique splicing factors by combining different PUF domains with effector modules that activate or suppress splicing. To prove the design concept, the researchers created six engineered splicing factors (ESFs) with distinct RNA target specificity. To determine if the ESFs could modulate alternative splicing of endogenous genes, they designed a specific RNA-binding module to target a gene that can either protect or kill cancer cells. They found that these ESFs could increase the amount of the splicing that promotes cell death and increases chemosensitivity of cancer cells.
"The potential to use this approach therapeutically is certainly intriguing," said William Schrader, Ph.D., deputy scientific director of NIEHS. "For starters, the method will have mostly investigational applications. But any cell-based method such as this provides in turn for assays that allow discovery of novel therapeutic drugs. The trick has been to find out how to target splicing events in specific ways, and this method is an advance along that path."
One of the next steps is to see whether this method can be used in mice to modulate alternative splicing. The researchers at UNC are currently using lentiviruses, or retroviruses, to express the protein in cells. Then, the researchers can use such factors in animal models of human cancers to test if they can promote tumor death in animals. The development of such gene-therapy tools will provide the means to test the technology in other organisms.
A patent on this technology has been filed. The NIH Office of Technology Transfer has also worked with the researchers to issue a licensing abstract on the technology. Those interested in collaborative research to further develop, evaluate or commercialize Modular and Artificial Splicing Factors should contact Suryanarayana Vepa, Ph.D., in the NIH Office of Technology Transfer.
Citation: Wang Y, Cheong CG, Tanaka Hall TM, Wang Z. (http://www.nature.com/nmeth/journal/v6/n11/abs/nmeth.1379.html) 2009. Engineering splicing factors with designed specificities. Nat Methods. [Epub ahead of print]
(Robin Mackar is the news director in the NIEHS Office of Communications and Public Liaison and a regular contributor to the Environmental Factor.)