By John S. MacNeil
It’s not all that often that biologists come across a completely new mechanism for gene regulation. Recently, however, microRNAs — short strands of RNA that the body uses to interfere with mRNA translation — have vaulted to the fore as one of the more significant means of determining which genes are expressed under certain circumstances.
With respect to basic molecular biology, microRNA research — that is, finding them, identifying which genes they regulate, and understanding their regulatory role in the grand scheme of things — is the exciting field du jour. In just the first two months of this year, two separate research groups reported that the reach of microRNA regulation extends to one-third of the protein-coding genes in the human genome. Given that just a year ago scientists knew of only 200-odd putative microRNAs, such a blistering pace of discovery means that only now are we beginning to have a clue what microRNAs are actually up to within the cell.
Some scientists — and, yes, a few companies — are hoping to understand and harness microRNAs for applications in medicine. But regardless of whether microRNAs turn out to be useful as biomarkers or aids in developing new therapeutics, the discovery of a new mechanism necessary for basic biological function is an occasion worthy of pause. While the discovery of microRNAs’ significance also points to how much we don’t know about basic biology, it’s a concrete step toward bridging that knowledge gap.
Of course, it’s clear that microRNAs are currently enjoying a popularity reserved for “the next big thing.” As has happened with many a biotechnology breakthrough, some researchers tend to overstress the potential significance of a finding, tool, or technique — think gene therapy — only to find that its application is more mundane. And it could be that the microRNA mechanism will ultimately only be interesting to a rarefied few scientists concerned with the arcane intricacies of gene regulation.
But chances are research around microRNAs will have some staying power. Gene regulation, after all, is a fundamental biological mechanism and the number of interested researchers is growing. A MedLine keyword search of “microRNA” turns up 20 papers published in January of this year, compared with eight in the first month of 2004. In addition to a growing number of academic scientists taking an interest in microRNA research, industry labs are also biting at the bait. Aside from siRNA therapeutics companies — like Alnylam and Merck through its Rosetta Inpharmatics subsidiary — Israeli company Rosetta Genomics has built a business around applying microRNAs as biomarkers and as tools in drug discovery and development (see box, p. 24).
The microRNA Mystery
The microRNA story actually begins back in 1993, when researchers led by Victor Ambros at Dartmouth University discovered a short RNA strand called lin4 that was essential for the timing of development in C. elegans. Ambros and his group concluded that lin4 did not code for a protein, but at the time no one knew what its real function could be; most researchers assumed it was an anomaly specific to C. elegans. Only later, in 2000, after Harvard Medical School geneticist Gary Ruvkun discovered another short RNA called let7, did scientists find that these short RNAs had homologs in frogs, polychaete worms, flies, mice, chickens, even humans. “We were working on [microRNAs] before we knew what they were,” says Ronald Plasterk, a molecular biologist at the Netherlands Institute of Developmental Biology in Utrecht.
But with the discovery of RNA interference, scientists now had another clue as to the actual function of these short RNAs. As it turned out, the RNAi mechanism that creates siRNAs, which uses an enzyme called Dicer to cut a precursor double-stranded RNA into pieces with 22 to 23 nucleotides, is quite similar to how the cell creates lin4. In fact, lin4 starts out as a 70-nucleotide piece of RNA that is also cleaved by Dicer.
So how exactly do siRNAs and microRNAs differ? In practice, siRNAs mark an mRNA strand for destruction by endonucleases, whereas microRNAs — with a few exceptions — merely prevent the mRNA from being translated into protein. The key lies in how microRNAs interact with the target mRNA strand. Unlike siRNAs, which exactly complement the target mRNA strand, microRNAs tend to line up imperfectly with the target sequence — enough to bind with the target, but not enough to mark them for annihilation. Instead, the slight mismatch between microRNA and its target is believed to cause a bulge in the microRNA-mRNA complex, which effectively prevents the mRNA from translation into protein. There are a few exceptions, however, with cases in which the microRNA does match up perfectly with its target, leading to a somewhat blurry distinction between microRNAs and siRNAs.
A perhaps more profound difference between siRNAs and microRNAs lies in how researchers currently understand their roles within the cell. RNAi, working through siRNAs, is understood to have evolved as a defense mechanism against RNA viruses — when the cell recognized foreign RNA, it would deploy siRNAs to mark it for death via endonucleases. MicroRNAs, on the other hand, are believed to function primarily as a means of regulating gene expression post-transcriptionally — that is, once the cell’s machinery has already transcribed a gene into mRNA.
Making the Connection
Scientists didn’t fully appreciate the significance of this process until very recently because there were only some 200 human genes known to encode for microRNAs. In January of this year, however, researchers at MIT and the Whitehead Institute used a computer algorithm to predict that microRNAs regulate more than one-third of all human genes. The algorithm, called TargetScan, which the researchers had validated with experimental work in a previous paper, uses a comparative genomics approach to predict which mRNA sequences are likely to be targeted by microRNAs.
“Built into TargetScan is a model for how microRNAs and their target mRNAs interact,” says Ben Lewis, the first author on the Cell paper along with Whitehead’s David Bartel and Christopher Burge at MIT. By comparing the frequency with which the telltale signs of this interaction appear across multiple genomes, including the human, mouse, rat, dog, and chicken, Lewis says he could predict the percentage of these interactions that are real. As a control, he also allowed TargetScan to gauge the frequency with which an arbitrary sequence was conserved across the various organisms’ genomes.
The result, somewhat startingly, was that more than a third of human genes are likely subjects of regulation by microRNAs. “We’re finding now that post-transcriptional regulation is underappreciated,” says Lewis, a graduate student researcher working with Bartel and Burge. “There’s a lot of evidence that post-transcriptional regulation using microRNAs is a lot more important than we previously anticipated.”
Lewis’ finding drew support from another paper, published online in Nature at the end of January, which described the use of microarrays to study the effects of two specific microRNAs on the expression profiles of cells in culture. Lee Lim, a computational biologist at Merck subsidiary Rosetta Inpharmatics, along with collaborators at the Whitehead Institute, report that the two microRNAs under investigation — miR-124 and miR-1 — regulate a far greater number of mRNA targets than they had expected.
Lim’s results also shed light on the type of cellular processes microRNAs are prone to regulate. Scientists already knew that certain microRNAs tend to be found only in specific types of tissue; in their experiments, Lim and his colleagues found that miR-1, which is normally found in muscle tissue, shifted the gene expression profile of the cells in culture toward that of muscle, and miR-124, normally found in the brain, shifted the cells’ gene expression profile toward that of the brain.
Ronald Plasterk, who spoke at a January Keystone Conference on gene regulation about his recent work, suggests that the regulatory role of microRNAs is more nuanced than might initially appear. His research, which involves studying microRNAs in situ with the aid of Exiqon’s Locked Nucleic Acid technology, supports the idea that microRNAs are organ specific, but don’t seem to be involved in early stage cell differentiation to create that organ or tissue type in the first place.
“What we have seen in these in situ [experiments] is that the expression at a very early stage seems to be weak — if at all existing — while once the organs are there you keep seeing the microRNAs,” Plasterk says. “[This suggests] that they’re not switchers, they’re not factors that tell a cell, ‘Hey, you have to become a liver cell, you have to become a neuron,’ but that the timing seems more consistent with the idea that they are to maintain something that has switched. To sort of remind the cell, ‘Remember who you are and where you came from. You’re a liver cell.’”
Given that microRNAs tend to pop up preferentially in certain organs or types of tissue, scientists also suspect they might have some degree of medical relevance. A recent report in Trends in Genetics, for example, explores a potential role for microRNAs in regulating the mechanism associated with Fragile X mental retardation. Similarly, another recently published paper in the Proceedings of the National Academies of Science discusses the potential role of microRNAs in regulating processes associated with leukemia. However, factoring microRNA regulation into the equation would seem only to add another level of complexity to understanding disease mechanisms, which could make developing therapeutic strategies even more difficult.
That said, there’s no doubt microRNAs will be an intense subject of inquiry for the foreseeable future. Even if the ramifications for understanding human disease are still a fair ways off, the insights into basic biology will be quite powerful, says Jim Carrington, whose work with microRNAs at Oregon State University has focused primarily on plants.
“I’m astounded at the pace of progress in understanding mechanisms and … how microRNAs integrate into developmental and other pathways,” Carrington says. “If you’re complacent in this field, you will be bypassed very rapidly. Not only is the pace of progress very rapid, but the interesting science that’s coming out is really quite unique.”
Eye on the Clinic
To be sure, microRNAs aren’t just the subject of academic research. Aside from companies engaged in developing therapeutics based on RNA interference, one Israeli company, Rosetta Genomics, has chosen microRNAs as the sole object of its attention.
Rosetta Genomics’ strategy, according to CEO Isaac Bentwich, is to integrate bioinformatics with microarray analysis to detect and make use of undiscovered microRNAs in medicine. Evidence in the literature suggests that microRNAs are associated with pathways involved in obesity, diabetes, cancer, and hematopoesis, Bentwich says, and he’s out to position his company to capitalize on their relevance to disease.
But Bentwich isn’t just talking about developing therapeutics on the basis of the insights from microRNA regulatory function. “We believe … microRNAs don’t just offer the ability to use [them] as the basis for therapeutics; but also in theranostics,” he says. “We aim to be a big player there.”
In the short term, he says, a microRNA-based biomarker could prove highly useful, both in diagnosing patients and in stratifying patient populations to help narrow the focus of a drug development program. In the long run, understanding microRNAs should prove advantageous in designing siRNAs for therapeutic applications, he says.
Researchers at Merck subsidiary Rosetta Inpharmatics (no relation to Rosetta Genomics) have also taken an interest in microRNA research, recently describing their work in a Nature publication. Meanwhile, siRNA therapeutics company Alnylam has secured IP rights to microRNAs discovered in Tom Tuschl’s lab at the Max Planck Institute, according to CEO John Maraganore. Another player to watch out for: Isis Pharmaceuticals, whose Singapore R&D group is developing antisense inhibitors and small molecules to interact with miRNAs relevant to diseases such as SARS and cancer. — JSM
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Calin, G.; Liu, C.; Sevignani, C.; et. al. "MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias" PNAS. 2004, 101, 11755-60.
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