October 24, 2013

Grad students help achieve key discovery

A multidisciplinary study conducted by the combined efforts of Vanderbilt University graduate students has led to the first evidence that abnormal messenger RNA export from the nucleus to the cytoplasm can cause human disease.

From left, Melanie Ohi, Ph.D., graduate students Scott Collier, Aditi and Andrew Folkman, and senior author Susan Wente, Ph.D., reported a new molecular disease mechanism in the journal Cell this week. (photo by Steve Green)

A multidisciplinary study conducted by the combined efforts of Vanderbilt University graduate students has led to the first evidence that abnormal messenger RNA export from the nucleus to the cytoplasm can cause human disease.

The discovery, reported in the current issue of the journal Cell, was made possible by applying genetic, imaging, biochemical and structural studies in two model systems — budding yeast and human cells, said senior author Susan Wente, Ph.D.

Thanks to the collaboration of structural and cell biology labs, the researchers were able to determine the key molecular event underlying a rare but lethal motor neuron disease, said Wente, associate vice chancellor for Research and senior associate dean of Biomedical Sciences.

“This study reflects the truly collaborative and collegial research environment and the amazing expertise we have across centers and departments,” she said.

The key player in this story is Gle1, a protein involved in gene regulation that Wente and her colleagues discovered in 1996.

Gle1 helps transport the transcribed copy of DNA, called messenger RNA or mRNA, from the nucleus to the cytoplasm, where it is translated into proteins. This transport from nucleus to cytoplasm takes place through the nuclear pore complex (NPC).

Wente, professor and former chair of the Department of Cell & Developmental Biology, has made major contributions to understanding NPC structure and function. In 2008, she and colleagues discovered that Gle1 not only helps transport mRNA; it is also crucial for translating mRNA into protein.

That same year, researchers in Finland linked mutations in the GLE1 gene to a rare human disease called LCCS1 (lethal congenital contracture syndrome 1), which leads to fetal death by 32 weeks of gestation.

Now the question was how? The answer seems to be in “oligomerization,” the ability of Gle1 to “oligomerize” or form disc-like complexes with itself specifically at the NPC.

Working with cell culture models of the disease and live cell microscopy, Andrew Folkmann, the study’s first author and a graduate student in Wente’s lab, found that the mutant Gle1 protein had altered movements in and out of the nucleus.

Folkmann also learned that Gle1 had a previously unreported ability to self-associate in cells. However, to solve the mystery, he sought out the expertise of the Center for Structural Biology’s Melanie Ohi, Ph.D., assistant professor of Cell & Developmental.

Using a technique called analytical ultracentrifugation, Folkmann detected Gle1 protein oligomers of a “higher-order” complexity than he’d expected.

Then he and Scott Collier, a graduate student in Ohi’s lab, teamed together to use purified proteins and single particle electron microscopy techniques to discover that the Gle1 oligomers formed disk-shaped particles in solution. And, the mutant protein was perturbed — forming abnormal-shaped discs.

“It’s really nice to be able to see something you’ve purified,” Ohi said. “That makes a big difference in how you might think about the problem.”

Aditi, another graduate student in the Wente lab, set up a “knockdown, add-back system” in cell culture that mimicked the human disease. With only the mutant protein in the system, the researchers showed the cells had defective mRNA export.

The study not only provided the first evidence that specific Gle1 defects in mRNA export can cause a lethal human disease, but suggested that altered mRNA export at the NPC is a new molecular disease mechanism.

“This is an outstanding example of how basic science discoveries can impact the understanding of human disease,” Wente concluded.

Xiaoyan Zhan, a research assistant III in the Wente lab, also contributed to the paper. The research was supported by National Institute of Health grants GM051219, NS070431, OD004483, CA119925, GM008320 and CA068485.