New understanding of microRNAs

Ribonucleic acid, or RNA, has long been thought to be important only to translate a gene's DNA into the proteins that are cells' workhorses. But new evidence shows that tiny bits of RNA not used to make proteins actually play central roles in normal biology and in the development of cancers.

"Scientists have known for a few years that production of these tiny RNAs, known as microRNAs, is only supposed to happen at certain times and in certain tissues, but no one had been able to identify what controlled the timing," says Joshua Mendell, M.D., Ph.D., assistant professor in the McKusick-Nathans Institute of Genetic Medicine. "We've identified the first such controller, a well-studied protein called Myc. Our discovery fits in quite well with the two other labs' studies on the involvement of microRNAs in cancer."

The work from investigators at Johns Hopkins is one of three papers on microRNAs in the June 9 issue of Nature.

Identified only a few years ago, microRNAs' best-known function is to control the extent to which other genes can be used to make proteins, by binding to and interfering with genes' protein building-instructions. The microRNAs play roles in cell division, cell specialization and cell death in worms and flies and are off-kilter in human cancers, but the Myc protein is the first factor identified that controls the production of microRNAs.

Myc (pronounced "mick") is already known to regulate about 10 percent to 15 percent of the genes in the human genome, controlling the extent to which they are used to make proteins. Myc also is faulty and overactive in many human cancer cells, although exactly how it contributes to cancer is unclear.

Given Myc's regulatory role and its involvement in cancer, the Hopkins researchers tested human cells to see whether changes in the amount of Myc affected levels of any of the more than 200 known microRNAs. Through these experiments and others, they discovered that Myc directly controls the gene for a set of six microRNAs in a region of chromosome 13 that is already tied to development of human lymphoma.

MicroRNAs are made by the same cellular machinery that makes other forms of RNA -- like the messenger RNA used to build proteins. However, microRNAs are immediately processed by cellular machinery that deals with what is known as RNA interference -- a biological phenomenon that specifically prevents RNA from being used to make proteins.

Accompanying the Hopkins paper are two reports looking specifically at microRNAs in cancer -- one in humans, one in mice. One report shows that human tumors' microRNA "fingerprints" -- patterns showing which of the more than 200 known microRNAs are more or less abundant than normal -- identify the tumors' tissue of origin much better than other tests. The other paper shows that over-expression of chromosome 13's microRNAs dramatically increases the risk of cancer in mice predisposed to cancer because of a faulty Myc gene.

"We've found that there's complex crosstalk between Myc, the microRNAs and the genes that both control," says Mendell. "This complex system, if disturbed, has the potential to very potently drive cell growth and cancerous proliferation."

Among the genes Myc controls is one called E2F1, which, like Myc, also controls the expression of genes. One of the genes controlled by E2F1 just happens to be the gene for Myc.

"This establishes a feedback loop," says Kathryn O'Donnell, Ph.D., who conducted much of the work as a graduate student in the Human Genetics Program. "Because both Myc and E2F1 increase the expression of genes that increase cell growth, the pair could be quite dangerous if they simply fed off each other."

But the researchers also discovered that two of the six microRNAs controlled by Myc actually reduce cells' ability to use E2F1's protein-building instructions. Essentially, these microRNAs act as a "brake" to slow E2F1's growth-promoting effects.

"So the Myc protein 'turns up' the genes for E2F1 and the microRNAs, but the microRNAs reduce the amount of E2F1 protein that can be made, fine-tuning Myc's effects, perhaps," says Mendell. "We chose to look for interactions with E2F1 specifically, but there are bound to be many, many more genes that these microRNAs regulate."

"Whether too much or too little of these or other microRNAs is a good thing or a bad thing -- whether it would contribute to or help prevent cancer -- depends on their targets in the cell," adds Myc specialist Chi Dang, M.D., Ph.D., the Johns Hopkins Family Professor in Oncology Research and a professor of medicine in the McKusick-Nathans Institute and the Johns Hopkins Kimmel Cancer Center. "Slowing E2F1 production would seem to be a good thing because doing so would slow cell growth, but that might not be the case for other genes controlled by these microRNAs."

Encoded for by genes' DNA, just like other RNA, microRNAs start out more than 1,000 building blocks long. Because they are able to fold back on themselves, they form double-stranded RNA, rather than the single strand characteristic of messenger RNA. As a result, the cell breaks the RNA into pieces. All sections except the microRNAs, just 21 to 23 building blocks long, is discarded.

The microRNAs can bind to single-strand messenger RNAs of the right sequence, which at the very least interferes with their ability to be read to make proteins and sometimes leads to the RNAs' destruction.

"Now we're figuring out the functions of these Myc-controlled microRNAs, other genes they regulate, and how else they might be involved in Myc-mediated initiation of cancer," says O'Donnell, who conducted her graduate work in Dang's laboratory.

The research was funded by the National Institutes of Health, the March of Dimes and a Sidney Kimmel Pilot Project Grant. Authors on the paper are O'Donnell, Dang, Mendell, Erik Wentzel and Karen Zeller, all of Johns Hopkins.