A study led by Dr. Geena Skariah, a recent Neuroscience graduate of the Ceman lab in Cell and Developmental Biology, and current postdoctoral researcher at the University of Michigan, revealed the importance of the protein Mov10 (Moloney leukemia virus 10) in neurological development in animals. The findings were published in BMC Biology.

The study began with an idea from fellow graduate student in the Ceman lab, Miri Kim. “Why don’t you look at the brain over time and see what happens with the protein Mov10?” she asked.

“It was a novel question because Mov10 has been characterized only in cell lines as an RNA helicase and as a regulator of retrotransposons,” said Dr. Stephanie Ceman. “No one was really looking at its effect on the brain.”

Skariah was intrigued. She walked over to the lab of Dr. Auinash Kalsotra, a biochemist who, along with Dr. Ceman, focuses on RNA biology. Skariah knew that the Kalsotra lab was working on cytosolic poly(A)-binding protein 1 (PABPC1) in the livers and hearts of mice and asked if they would be willing to share resources.

“We both share the interest in how RNA brings things together and makes the cells and tissues in our bodies work,” said Dr. Kalsotra.

From the resources available in the Kalsotra lab, Skariah was able to test post-mortem brains of mice at different points in their development and measure the level of Mov10.

“We saw that Mov10 goes up during development and goes back down in the adult,” said Skariah. “That was my first piece of real data.”

No one had ever looked at Mov10’s role in the development of the brain, and this was a completely new finding. “That finding started off this whole project,” said Skariah.

Mov10 was thought to be unimportant to brain function because there are usually low levels of it found in adult brain.

“I didn’t think they were going to see anything. I didn’t say not to do it, but I did say it was not going to work,” admitted Dr. Ceman, with a laugh.

But Skariah was able to convince Dr. Ceman with the impact of her first results. “That’s great!” Ceman said. “Now test it in different sexes and with different strains.”

“Every single day. Over and over and over!” quipped Dr. Kalsotra.

The initial finding opened the door to a cascade of questions. Why is this protein going up in early development? What is it is doing in the brain?

“What’s fun about this,” said Ceman, “it’s the biggest discovery project I have been part of in a long time.” Every time they did an experiment in this project, they had no idea what they were going to find.

Further experiments showed that reduced levels of the protein resulted in behavioral changes that suggested neurological impairment. They were seeing several defects in the cells, but where were they coming from? To address this question, they moved to a neuroblastoma cell line (Neuro2A), on which they could use CRISPR-Cas9 to remove both copies of Mov10. Then they examined the effect of Mov10 loss on total RNA levels and on the ability to grow a neurite, Ceman explained.

“They sequenced the cellular RNA millions of times at the Carver Biotech Center on campus and obtained a huge amount of data showing a lot of differences in the RNA levels,” said Kalsotra. “We all started to talk about what to make of it and delve into the data.”

The Ceman and Kalsotra labs have an RNA journal club that meets every week, and through this club, Skariah learned that Joseph Seimetz, in the Kalsotra lab, could help with the bioinformatics on this project.

“There is a list of gene candidates, and you can’t test every one because there are thousands of them,” said Skariah. “Bioinformatics is so powerful because you can pick important ones and remove them from the cell.”

“The Ceman lab basically did all the work,” said Seimetz. “It was perfect timing. We had started to build our own bioinformatics pipelines for analyzing “Big Data,” so we combined it with our work.”

“Joe developed the graphics that were so important for the paper,” said Skariah. “We could move from a point of taking out genes to a point where we see a phenotype, we remove it and see how the genes change and how they function.”

There is a lot more research that can come from this data. Their next step is to see what happens when Mov10 is removed from the brain. The lab is continuing to look at how the reduction of Mov10 changes the neurons. The findings were published in BMC Biology, and the paper has piqued significant interest, with nearly 5000 views in the last six months.

Seimetz’s work organizing the sizable amounts of data collected during the project is now shared in a public database that allows scientists around the world to work with and use the data.

“It’s like our data keep on giving,” Dr. Ceman said.

Story Source(s)