Genetic screening in yeast reveals new candidate gene for amyotrophic lateral sclerosis

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Nov 15, 2011

Mutations in 2 related proteins, TDP-43 and FUS, cause some forms of amyotrophic lateral sclerosis. Specifically, these 2 proteins are RNA-binding proteins that connect to RNA to regulate the translation of proteins and other cellular functions such as RNA splicing and editing. In a new study, researchers at the Perelman School of Medicine at the University of Pennsylvania discovered additional human genes with properties similar to TDP-43 and FUS that might also contribute to amyotrophic lateral sclerosis.

There are over 200 human RNA-binding proteins, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to amyotrophic lateral sclerosis pathology. Using yeast as a model organism, the lab of cell biologist and senior author Aaron Gitler PhD, assistant professor of Cell and Developmental Biology, surveyed 133 of these proteins. They further winnowed the candidates using bioinformatics to about 10 proteins, selecting one called TAF15, to study further. Their findings appear this week in the Proceedings of the National Academy of Sciences.

A group of high school students performed the screening in the yeast cells in the Gitler lab during the summer of 2009 with the aim to more rapidly locate disease genes associated with amyotrophic lateral sclerosis . They were from Philadelphia area schools - J R Masterman Laboratory and Preparation School, Central Bucks High School East, Radnor High School, and Conestoga High School - and most of the students have gone on to pre-medical programs or biology majors in college.

"We used yeast to predict new ALS disease genes," explains Gitler. "TDP-43 and FUS are both RNA-binding proteins involved in ALS and we have found previously that they both aggregate and are toxic in yeast. We wondered if there were more proteins like these out there and, if so, we probably could find them using our yeast screening approach. We performed a large screen for other human RNA-binding proteins with properties similar to TDP-43 and FUS in yeast and then validated one by sequencing the DNA of ALS patients. We also did experiments in cells and fruit flies to see how the new protein functioned."

From DNA derived from 735 sporadic amyotrophic lateral sclerosis patients and over 1300 controls, the team found mutations in TAF15 that were absent in healthy controls. These variants, when expressed in cultures of rat embryo spinal cord neurons, caused the protein to clump in the cytoplasm of the neurons, similar to clumping seen with TDP-43 and FUS. What's more, working with Nancy Bonini PhD, a Penn professor of Biology, they found that TAF15 initiated neurodegeneration in fruit flies. Bonini is also an Investigator of the Howard Hughes Medical Institute.

The lab of James Shorter PhD, assistant professor of Biochemistry and Biophysics, provided more clues about TAF15. In an aggregation assay using purified TAF15 protein, they found that mutant versions of TAF15 clumped faster. The purified TAF15 protein also harbored a prion-like portion in its amino acid sequence, much like TDP-43 and FUS, which might be a future predictor of neurodegenerative qualities in mutant proteins.

The lab of Zissimos Mourelatos MD, associate professor of Pathology and Laboratory Medicine, tested effects of TAF15 in cell cultures of neurons derived from mouse embryonic stem cells and found that TAF15 mutant versions accumulated abnormally in the cytoplasm of these cells.

Finally, looking at postmortem samples from sporadic amyotrophic lateral sclerosis patients in the tissue bank maintained by co-authors John Trojanowski MD PhD, director of the Penn Institute on Aging, and Virginia Lee PhD, director of the Center for Neurodegenerative Disease Research at Penn, the team found clumps of TAF15 accumulated in human spinal cord neurons in these amyotrophic lateral sclerosis patients.

Taking all of these lines of evidence into account, the authors propose that aggregation of RNA-binding proteins might contribute very broadly to amyotrophic lateral sclerosis pathology. Looking forward, Gitler hopes that, “the genes identified in the yeast screen, coupled with prion-like domain prediction analysis, will provide a powerful resource to speed ALS disease gene discovery.”

This research was supported in part by the National Institute of Neurological Disorders and Stroke.

Source: News Release
Penn Medicine
November 15, 2011