Silencing Genes with Non-Coding RNAs

By Jeff Worley

There are as many roads that lead to a life in the lab as there are scientists, but Kevin Morris's path was a particularly unusual one. "For starters, I was a lousy student in high school, usually at the bottom of whatever class I was in, and I never gave science a thought." But something happened in 1986 that changed Morris's life dramatically. That "something" was the headline-grabbing spread of HIV.

"My imagination was captivated by the idea that a virus—something you can't see—can kill people. How did this killer operate? And what could we do to stop it?"

Morris, now an assistant professor of molecular and experimental medicine at the Scripps Research Institute who was recently named one of "Tomorrow's PIs" by Genome Technology Magazine, caught fire in his high school molecular biology class, going from worst to first. This sudden ascendency was met with skepticism by his teacher. "On the mid-term, after I'd made the highest score, he said, 'I think you cheated on this, but I just can't figure out how," Morris recalls, laughing.

As an undergraduate student focusing on wildlife and ecology at Humboldt State, he continued to be intrigued by the HIV virus, an intense interest that led him to the University of California, Davis, and a Ph.D. in 2001 in comparative pathology, microbiology, and immunology. Morris did postdoctoral research in this field at the University of California, San Diego (UCSD), and then worked as a research scientist at the Beckman Research Institute at the City of Hope before coming to Scripps Research in 2005.

Morris's current research here is a continuation of his work in a gene therapy lab at UCSD, where he got in on the "ground floor" of the scientific community's new focus on RNA interference (RNAi). Also known as RNA silencing, RNAi is the introduction of double-stranded RNA into a cell to inhibit the expression of a gene. "In our lab, we started asking whether we could use small interfering RNA to target gene promoters and turn the gene off at that level—transcriptional gene silencing," says Morris. "And we accomplished this." He continued this work at the City of Hope, targeting the mechanism of RNAi.

"Over the past few years it has become increasingly apparent that many RNA-mediated modes of gene regulation are operative in biological systems," Morris explains. "Now scientists are beginning to understand just how pervasive this network is and to what extent it may be possible to apply this phenomenon for therapeutic benefit." Adding to the complexity of this regulatory network is the recent observation Morris made in his work at the Beckman Research Institute, and research by two other groups in the field, that small non-coding RNA (ncRNA)-mediated transcriptional regulation can act in both a suppressive and activating manner in human cells.

Non-coding RNAs, the focus and trigger of Morris's current work, are RNA molecules that don't code for a protein. And since they don't translate into protein, scientists have long wondered what they are they good for, shoveling them, meanwhile, into the box labeled "Junk DNA." This question served as the starting point in Morris's current work, funded by an RO1 grant and the Stein Endowment Fund at Scripps Research, which resulted in an article published November in PLoS Genetics titled "Biodirectional Transcription Directs Both Transcriptional Gene Activation and Suppression in Human Cells."

Morris describes the lab work that led to these findings as "solid but unspectacular science in action." With graduate students Peter Hawkins and Anne-Marie Turner, Morris grows cells in a culture and puts them into petri dishes. The researchers then mix up a "transfectionary agent cocktail" made of lipids and small non-coding RNAs, and treat the cells with this mixture. The cells are left alone for 48 to 72 hours; then the team does an assay, reading out the results in DNA and RNA.

"It doesn't look like we're doing much besides pipetting water from one place to another," Morris admits. "It's not dramatic—the dramatic aspect of the work is the questions we're asking and the unique answers we've gotten."

Results

In the PLoS Genetics article, Morris shows the mechanism whereby small interfering RNAs, when put into the cell, cause a specific upregulation in expression of the targeted gene. The researchers discovered that the RNAs targeted a RNA-based suppressor, and that this action resulted in a loss of suppressor and turned up the specific gene.

"This was a really a big discovery because the RNA-based suppressor was a non-coding RNA, which is thought to make up around 98 percent of the genome," Morris says elatedly. "Our paper suggests some answers, with a few concrete examples, as to what the 98 percent of the genome is doing." This discovery redefines the thought that this part of the genome is merely "junk DNA." Morris's team showed that this RNA-based suppressor is functional in regulating gene expression. The manner by which it regulates gene expression—through directed epigenetic changes—correlates well with a mode for ongoing natural selection and evolution, Morris adds. An epigenetic change affects a cell without directly affecting its DNA. Overall, these data suggest that non-coding RNAs regulate gene expression and also function to assist the cell in responding to selective pressures, for example, how the cells Morris works with evolve different gene expression patterns to overcome the stresses and strains of life.

So what's the human benefit of this work?

"In terms of basic science, it's very cool to see how cells undergo evolution and selection," says Morris. "Because these non-coding RNAs are active at controlling the gene promoter, the genome is always undergoing changes. We've shown how non-coding RNAs function."

Beyond the basic increase in scientific knowledge, Morris says that the potential therapeutic benefits are enormous.

"If you can find a gene that's specifically linked to a particular disease, BCRA is a good example, through this process you can theoretically turn this gene off." Inheriting a mutation in either the BRCA1 or BRCA2 gene is a hereditary predisposition for breast cancer. The CTCF gene is another example of a culprit gene that could be switched off. It encodes for a transcriptional repressor of the c-myc oncogene and has been linked to invasive breast cancer. Morris clarifies the point that so far in his lab, genes of interest haven't been turned off, but have been "turned down" by as much as 80 percent. "The main point is, we've shown how to manipulate a gene's function and power."

"This is an important study that provides new mechanistic insight into the recently described phenomenon of transcriptional gene activation by small RNAs," says John Rossi, a professor in the Division of Molecular Biology and dean of the Graduate School of Biological Sciences at the Beckman Research Institute "What is particularly significant is that the siRNAs are triggering transcriptional activation by post-transcriptionally directing cleavage of an RNA that traverses the promoter region in an antisense orientation, and in the presence of a promoter sense transcript, triggers heterochromatic silencing of that promoter." "Antisense" is the strand opposite to that which expresses a protein-coding transcript (mRNA) or another non-coding RNA. "These studies have important implications for understanding how tumor suppressors might be inactivated in cancers by imbalances of promoter traversing antisense transcripts."

Send comments to: mikaono[at]scripps.edu

"Our [latest] paper suggests some answers, with a few concrete examples, as to what the 98 percent of the genome is doing," says Assistant Professor Kevin Morris.