It’s a Small World

As MicroRNA List Grows, So Does Genomic Complexity.

Look up the word “gene” in the dictionary, and you will more than likely find a definition that goes something like this: A gene is the basic unit of inheritance found at a specific location along a chromosome. Each individual gene in the genome encodes a particular protein.

If only it were really that simple. For the last couple of decades, scientists have known there is another sort of gene altogether – sequences of DNA that are transcribed into RNA, as all good genes are, but then don’t get translated into proteins. There are in fact many thousands of these noncoding RNAs, and they come in various flavors in the genome, some quite large, but others very small.

“It appears to us there is a whole world out there of small RNAs that we can imagine playing all kinds of different roles no one has even thought to consider before,” says Sandeep Dave, an IGSP Investigator and Assistant Professor in the Department of Medicine.

Perhaps the best known among those noncoding RNAs are the rather tiny but influential microRNAs, first discovered in the experimental worm C. elegans. At just 18 to 22 “letters” long, those microRNAs are in some ways more anti-gene than gene. They silence other genes by binding RNA messages and preventing their translation into proteins.

“Ten years ago, we thought microRNA was a minor, esoteric aspect of C. elegans development,” says Philip Benfey, Director of the IGSP Center for Systems Biology. “Through genome sequencing efforts and the comparison of genomes, there’s been an explosion of small RNA discovery and a realization that there is this whole other layer of gene regulation involving very small RNA molecules that had been overlooked for more than 40 years. In science, that’s what happens: you believe something has been explained and then you find a whole new aspect.”

Prepare for Takeoff

Even though scientists discovered the first microRNA some time ago, it has really only been in the last few years that they’ve begun to appreciate what a deep role the tiny molecules play in biology. MicroRNAs have turned up in just about every place that anyone has looked for them. They have been implicated in everything from stem cell biology to the development of organisms to human disease.

But there is still an awful lot we don’t know. For starters, no one knows how many microRNAs exist in our own genomes or in any other genome, and even less about what they do and how that might influence health or disease.

In the early days, researchers were reporting microRNAs a small handful at a time, Dave says. In 2007, the tally of human microRNAs was a few hundred or so. Every couple of months, someone would uncover a couple more and the list would grow.

“Last year, we went from about 400 human microRNAs to over 1,000,” Dave said. “What that meant for people who study microRNA was constant obsolescence of the laboratory platforms used to measure their expression.” The universe of unknown microRNAs was almost as big as the known one and, as a result, the tools used to study them were constantly becoming outdated as new discoveries were made.

“It appears to us there is a whole world out there of small RNAs that we can imagine playing all kinds of different roles no one has even thought to consider before.”
—Sandeep Dave

A Deep Dive

Since the advent of next-generation sequencing tools, it’s become cheaper and easy enough for Duke researchers and others to take a deep dive into their favorite cells, sequencing all of the RNA within them – including the tiniest of RNA molecules – in an effort to finally nail down an exhaustive list of all the candidate microRNAs.

In a recent report, Dave and colleagues in the Hematologic Malignancies Research Consortium did just that in both normal and cancerous human B cells, blood cells that are the source for many increasingly common leukemias and lymphomas. Dave, who is an oncologist as well as a researcher, had earlier shown that microRNAs are key at every step of the way in those blood cells, making them potentially useful as biomarkers for distinguishing among different forms of blood cancers.

“Until we know what all the microRNAs are, we can’t get a complete handle on their functions,” Dave said. With that in mind, Dave team sequenced 31 normal and malignant human B cell samples, turning up an impressive 333 known microRNAs, more than twice the number that had ever been seen in any one type of tissue before. On top of that, they found 286 brand new microRNAs.

“Our work suggests that over a third of the microRNAs present in most cell types are currently unknown and that these microRNAs may regulate important cellular functions,” Dave says. Already, there are clues that some of the new microRNAs may be important in the process that turns healthy B cells into cancerous ones. Six of the new microRNAs they uncovered are encoded in a single cluster and appear to regulate a pathway with well-known links to cancer.

But Dave doesn’t think what they’ve found is relevant only in the case of B cells. Some of the microRNAs they found in their cells have been seen before in other kinds of cells at levels up to 100 times higher. “The majority are likely to have more far-reaching and broader functions,” he says. Sorting it all out will be enough to keep researchers occupied for decades to come.

An Unexpected Find

Meanwhile, IGSP Investigator Ashley Chi in the Department of Molecular Genetics and Microbiology has been unraveling the mysteries of microRNA in a very different type of human blood cell – one that might seem like the last place you’d expect to find microRNA.

Most textbooks will tell you that red blood cells – the oxygen-carrying component of human blood – have no genetic material of their own. Long thought to be passive containers for hemoglobin, red blood cells lose their nucleus along with other standard components of a cell as they develop, and that’s where most of a cell’s genetic material is housed.

Not long ago, Chi and his group made the surprising discovery that red blood cells contain microRNAs, and lots of them. In fact, Chi now describes the cells as “big bags of microRNA.” Because red blood cells lack the cellular machinery to make protein, however, it wasn’t initially clear what, if anything, these tiny RNAs might be doing in the cell.

“A lot of skeptics would have said this is simply left over junk,” says Chi.

In search of more clues, graduate students in the Chi lab decided to turn to another type of red blood cell: sickle cells. Sickle cell disease is caused by a genetic mutation that deforms red blood cells from their normal round shape into a rigid crescent, or sickle shape. These abnormally-shaped red blood cells can clog blood vessels, causing excruciating pain.

Sure enough, Chi’s group found that the microRNA profile of sickled red blood cells was vastly different from that of healthy red cells. The findings suggested to them that variation among red cells in microRNAs might hold answers to long-standing and rather perplexing questions about sickle cell and other blood diseases, for instance, why people who carry exactly the same sickle cell mutation can still show wide differences in the severity of their disease and its manifestations.

As evidence in support of that notion, Chi and his colleagues have since found that some people with more severe anemia as a result of sickle cell disease also have higher levels of a particular microRNA in their red blood cells. The microRNA in question – designated as miR-144 – controls a gene important to the cells’ ability to protect themselves against damaging free radicals. When miR-144 levels go up, the activity of the protective gene goes down and red cells are less likely to survive under stress, explaining the worse outcomes for those patients.

Since their initial discovery of microRNA in red cells, Chi’s group has also been toying with another tantalizing idea: microRNA might explain a mysterious advantage of sickle cell disease – that people who inherit the mutation are more likely to survive malaria. After all, they have noted, human microRNAs – including one that turned up at very high levels in sickled red blood cells in their analysis – had already been found in the Plasmodium parasite responsible for malaria, according to earlier reports.

With the final verdict still out on that front, Chi’s team has taken up the same sort of deep sequencing work that Dave’s group applied to B cells in an effort to find every last microRNA in the red cells. Their preliminary analysis suggests that they may uncover more than 50 new microRNAs in the cells, some of which appear to be unique to humans and other primates.

Going Green

MicroRNAs aren’t limited to the animal kingdom; plants have them too, if in smaller numbers.

“It appears animals use microRNA more than plants,” Benfey said. There are thousands of microRNAs in animals compared to hundreds in plants. That might change as scientists learn more, Benfey says, but he thinks it’s unlikely.

In their first foray into next-generation sequencing, Benfey’s lab in the Center for Systems Biology is searching for every last microRNA within the roots of the plant Arabidopsis thaliana, a species that is a staple of developmental and molecular biology studies. Key in their approach is what IGSP Investigator Uwe Ohler, a computational biologist and collaborator on the research, calls Benfey’s “cell sorting magic.”

They separate each individual cell type found in the root and deep sequence each of those independently. “If you took the whole root or flower, you would miss many microRNAs,” Ohler says, as the small strands of RNA primarily found in less abundant cell types would simply get swamped out.

Ohler’s group has devised new analytical methods to identify microRNAs in the Arabidopsis sequence data, based on some of the properties of microRNA that are specific to plants. Although the analysis is still in the works, both Benfey and Ohler are confident that they will ultimately up the number of known plant microRNAs by 20 to 30 percent or so.

Come On, Feel the Noise

As these lists of microRNA continue to grow, the challenge will be sorting out which ones really count.

“The trick with these new, high-throughput experiments is that if you see everything, you will find things that have no relevance,” Ohler says. “It might just be background transcription of small RNAs that have no target. If it doesn’t do anything, it doesn’t matter.”

Greg Wray, director of IGSP efforts in evolutionary genomics, seconds that notion. His group hasn’t focused in on the tiny microRNAs specifically, but early this year, they did find a great deal of variation in the presence of longer noncoding RNAs in the brains of humans, chimps and macaques.

“We saw a lot more noncoding RNA than we expected,” he said. “The question is whether they all have biological meaning or whether they just represent random firing of polymerase,” the enzyme that copies DNA into RNA.

One way to sort that out is to study each candidate gene, RNA by RNA, in search of a function. That’s a task that many scientists in the small RNA field are now working on, but it’s going to be slow going. Efforts by computational experts like Ohler can help, by developing tools that can accurately predict target genes from sequence data alone and that can begin to place those small RNAs into the larger context of regulatory networks.

Wray says that evolution can also be a guide. Their studies in primate brains showed that some of those noncoding RNA transcripts were conserved, both in terms of their location and expression, among humans, chimps and macaques. “It suggests they may have an unknown functional role,” said Courtney Babbitt, a postdoctoral researcher in Wray’s lab. The same evolutionary principles applied in that work can help to narrow down the growing lists of microRNA to those most likely to hold real biological significance.

Dave concedes that deep sequencing is especially powerful in turning up microRNAs that are present at very low levels in cells. But, he adds, even those seemingly bit players could turn out to be critical. After all, no one really knows how much of a given microRNA it takes to make a real difference. In some cases, he says, “a few copies of microRNA in a cell may be adequate to exert profound down-stream effects.”

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