Research in the Innerarity laboratory concerned a protein, apolipoprotein (apo) B. ApoB is found in two forms with very different functions in lipoprotein metabolism. Both are encoded by the same gene. One form, apoB100, is translated from the full-length mRNA, but the other, apoB48, is translated from a shorter piece of the same mRNA.
Dr. Yamanaka set out to define the mechanism responsible for the two forms. He investigated an enzyme that “edited” the apoB mRNA, called apoB mRNA-editing enzyme catalytic polypeptide 1 or APOBEC-1. In the liver, the mRNA is translated into a full-length protein, apoB100. In the intestine, the enzyme edited the apoB mRNA by deamination apoB nucleotide-6666 from a genomically encoded cytidine to a uridine. This modification changes the information in codon-2153 from glutamine to a stop signal, resulting in premature termination of translation and the formation of apo-B48.
This was an intriguing finding. Dr. Yamanaka hypothesized that overexpressing APOBEC-1 in the liver might be a way to decrease apoB100, the principal protein of “bad” cholesterol—LDL—which would lower the risk of heart disease. To explore this hypothesis, he generated transgenic rabbits and mice that expressed APOBEC-1 in their livers. In fact, the transgenic animals had lower levels of apo-B100 and LDL than control animals. However, unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed liver cancer.
What might have been a disappointing result for many scientists only served to whet Dr. Yamanaka's intellectual curiosity. What could explain this unpredicted result? Dr. Yamanaka hypothesized that the overexpression of the editing enzyme might affect other important mRNAs, and he decided to look for them. He found that a novel mRNA, which he called “novel APOBEC-1 target 1” or NAT1, was also aberrantly hyperedited, which also led to development of liver dysplasia and hepatocellular carcinomas. He was intrigued with this new protein.
“Our philosophy in bringing scientists like Shinya Yamanaka to Gladstone is to give them the freedom to follow wherever their curiosity and the science might lead,” said Gladstone president Robert W. Mahley. “He wasn't limited in his scope to just one lab, but he moved among the investigators throughout the institute to learn whatever he needed to know to advance his science.”
Dr. Yamanaka took full advantage of that opportunity.
“I decided to make mice that lacked the mouse NAT1 gene,” he said. “To make knockout mice, we needed embryonic stem (ES) cells. I asked my Gladstone colleague Bob Farese to teach me how to culture ES cells, how to do gene targeting, and how to perform micro-injections.”
The NAT1-/- ES cells seemed to be normal by most measures. They looked normal and had the expected gene expression profiles. However, they had lost the ability to differentiate. This finding showed that NAT1 controls specific gene expression pathways that are required for cellular differentiation.
“So from that very simple experiment, we learned that NAT1 is indispensible for pluripotency of mouse ES cells. Without NAT1, ES cells cannot differentiate, but they can still proliferate,” he said. “Until that finding, ES cells were just a research tool for me to use to make knockout mice, but because of that finding with NAT1, ES cells became my research targets, not tools.”
Since that time, Dr. Yamanaka has continued to study how cells differentiate from ES cells to adult cells. That research culminated in his groundbreaking work showing that adult cells can be reprogrammed to a ES cell-like state.
“Personally, I could be no more pleased with the success that Shinya has had,” said Dr. Mahley. “He has taken on a very difficult project, a project which most people would simply not undertake. It was far too risky, but he did have a vision. It was a big and a bold vision, and he was willing to work to accomplish this.”
[Note: ApoB mRNA editing is the first known example of RNA editing and the first example of RNA editing in mammals. Since this original report, several other examples of RNA editing have been found. In addition, the family of APOBEC enzymes has been shown to have significant anti-HIV activity and may represent a new family of anti-HIV drugs.]
