VICTOR AMBROS, PH.D. AND GARY RUVKUN, PH.D.

scientific_american

By John Rennie, Science Editor & Journalist

In fiction, scientific breakthroughs are always dramatic. When researchers make a paradigm-shattering discovery, the world instantly hails it as revolutionary. Real life can be slower to hand out rewards. After molecular biologists Victor Ambros and Gary Ruvkun reported on a peculiar exception to the normal rules of gene regulation, it took seven years for the significance of that finding to emerge.

Today, the anomalies discovered by Ambros and Ruvkun and called microRNAs are recognized as the first members of an unexpected world of tiny regulatory molecules that now number in the thousands. These short, single-stranded RNA molecules are about ten times smaller than anyone had thought to look for before Ambros and Ruvkun. They help control a wide variety of processes, both normal and pathological, including embryonic development, differentiation of blood cells, muscle function, congenital heart disease, viral infections, and cancers such as lymphoma and leukemia. Within just a few years, microRNAs have gone from a newly revealed feature of basic biology to a foundation for emerging medical practice.

In recognition of the conceptual and clinical importance of microRNAs, Ambros and Ruvkun have been showered with honors, including Albert Lasker Award for Basic Medical Research, the Gairdner International Award, the Breakthrough Prize in Life Sciences, the Gruber Genetics Prize and, in 2012, the Dr. Paul Janssen Award for Biomedical Research. These acknowledgements are well-deserved and recognize a long-term partnership between the scientists, which started in a laboratory at the Massachusetts Institute of Technology nearly four decades ago.

When Victor Ambros applied to M.I.T. for undergraduate study in 1971, his admission essay was just six words: “I want to be a scientist.” As a boy growing up in Vermont, he had read the biographies of famous researchers avidly, and they spurred in him a desire to become part of that historical tapestry of discovery and invention. Originally, he leaned toward a career in astronomy, in interest his parents had helped along with a kit for building a telescope.But it was biology that Ambros ultimately studied at M.I.T., both during his undergraduate years and then as a graduate student.
Under the Nobel laureate David Baltimore, Ambros worked on the structure of the polio genome. Then, in 1979 he joined the M.I.T. laboratory of Nobel laureate Robert Horvitz as a post-doctoral fellow, where he investigated the genetic regulation of development in the reproductive system of the roundworm Caenorhabditis elegans. (C. elegans has been studied in minute detail for decades, and is still a commonly used animal model for developmental biology and neurobiology research.)

During his work on C. elegans, Ambros uncovered an intriguing relationship between two of its genes, lin-4 and lin-14. Their effects were complementary: mutant worms in which the lin-4 gene was broken or underactive showed the same developmental defects as mutant worms in which the lin-14 gene was overactive. The logical conclusion to draw was that lin-4 normally suppressed the activity of lin-14.

Yet, it was not obvious how to isolate the lin-4 or the lin-14 genes to decode how they worked. To solve the problem, Ambros began to brainstorm with another newcomer to the lab, molecular biologist Gary Ruvkun.

Ruvkun’s journey to that laboratory was more circuitous. Born in 1952 in Oakland, Calif., he became electrified by astronomy, the first NASA spaceflights, and science in general. Like Ambros, he read voraciously and had parents who nourished his passions, with gifts of a telescope and microscope. At the University of California at Berkeley, Ruvkun became fascinated with physics, learning of revolutions on atomic and galactic scales. He also began to learn molecular biology, taking inspiration from the discovery of the double helix and genetic code. He graduated with a bachelor degree in biophysics in 1973.

After a couple of years of working in reforestation in Oregon and traveling throughout Latin America, Ruvkun returned to academia at Harvard in 1976. He took up the study of recombinant DNA technology in the laboratory of Frederick Ausubel, who was interested in engineering crop plants with the nitrogen-fixing ability found in some bacteria. In his PhD research, Ruvkun identified the genes that fix nitrogen in these bacteria and discovered that they are conserved over vast evolutionary distances. His focus on evolutionary conservation is a recurrent theme in his microRNA research as well. After earning his doctorate, Ruvkun also did research in Nobel laureate Walter Gilbert’s lab at Harvard, and brought his molecular genetics expertise to the Horvitz lab at M.I.T. In the Horvitz lab, Ruvkun and Ambros developed an approach to allow the molecular characterization of C. elegans genes, including lin-14 and lin-4.

Ruvkun and Ambros teamed up to investigate the interaction of lin-4 and lin-14, but by 1984, when they were each leaving to start their own labs—Ambros at Harvard and Ruvkun at Massachusetts General Hospital and Harvard Medical School—they had not yet found the answer. They agreed to keep working on the problem, with Ambros focusing on lin-4 and Ruvkun on lin-14. Cloning and characterizing the lin-4 gene was a painstaking process.

Ambros credits the arrival of Rosalind Lee in his lab as a technician in 1987 as a turning point. Lee (who had also worked in the Baltimore lab and had married Ambros in 1976) could devote the time and effort to the tedious job of cloning lin-4. She and post-doctoral fellow Rhonda Feinbaum worked for the next four years on the project, until they had finally identified lin-4’s sequence. Contrary to expectations that lin-4 might encode a small regulatory protein, they found instead that lin-4 makes a noncoding string of RNA only 22 nucleotides long, which in its precursor form folds back on itself like a hairpin.

During this same period, Ruvkun determined that the part of the lin-14 gene that is negatively regulated by lin-4 mapped to a region of the messenger RNA that is not translated into protein. And he discovered that this region was highly conserved in evolution.

As the exact RNA sequences of lin-4 and lin-14 emerged in the Ambros and Ruvkun labs, Ambros and Ruvkun decided to compare the sequences of their two genes to see if the model that these RNAs base paired with each other would be supported.

One evening, they read off the sequences of nucleotides to each other on the telephone and were astonished to see that portions of lin-4 and lin-14 were complementary, but not precisely complementary—there were surprising loops and bulges in the RNA duplexes, but duplexes nevertheless—a fact that suggested an amazing, unorthodox possibility."

Years earlier, molecular biologists had observed that some plants and bacteria fight off viral infections by deploying single-strand RNA molecules against them. These RNAs are described as antisense because they are complementary to sense sequences in the viral genes. By binding to viral gene transcripts, these RNAs somehow blocked the production of viral proteins.
Ambros and Ruvkun realized that something similar, but distinct with its bulges and loops and incredibly tiny length, could be going on between lin-4 and lin-14: very small RNA transcripts of lin-4 could be suppressing the expression of the lin-14 gene. The evolutionary conservation of both the lin-4 and the lin-14 RNA segments pointed to this base pairing, as well as the RNA bulges and loops, as being under evolutionary selection. Biotechnologists were already experimenting with using antisense RNA to shut down genes artificially. Perhaps nature had evolved some similar mechanism for silencing genes in a complex cell, too. And perhaps the design of perfect RNA duplexes was too simplistic and too long; nature had simply evolved more intricate and interesting forms of regulatory RNAs that Ambros and Ruvkun were the first to see.

After confirming their suspicion, Ambros and Ruvkun each wrote a paper about their discovery, and those papers were published back-to-back in the journal Cell in 1993.

Remarkable as this news was and despite the prominence of its publication, other scientists’ response to it was muted. Even Ambros and Ruvkun were unsure whether this bizarre regulatory mechanism was anything more than an oddity of the worm. As Ambros reflected in his acceptance speech for the Lasker Award (subsequently published as an essay for Nature Medicine in 2008), “Why was I so pessimistic about the prospect of lin-4 being the harbinger of a diverse class of regulatory molecules of broad importance? … There was no theoretical need to explain existing phenomena in terms of new mechanisms or new classes of molecules.” The traditional model of gene regulation involving protein transcription factors generally still worked fine.

Worse, Ambros and Ruvkun could not find equivalents to the lin-4/lin-14 microRNA system in other species distant from roundworms, so the mechanism might be irrelevant to other organisms. “Despite decades of model system genetics and gene cloning, no other example of a small RNA gene product like lin-4 had been identified. So, maybe lin-4 really was a peculiarity of Caenorhabditis developmental timing mechanisms,” Ambros wrote. However, Ruvkun observed in his acceptance speech for the Lasker Award (also published in Nature Medicine 2008), that there were precedents from RNA biology, for example, the ribosomal RNA, that are universal to all life, so that the lin-4/lin-14 paradigm could be general.

In 2000, miRNAs emerged from their corner of biology. Ruvkun discovered a second microRNA in C. elegans, let-7.

He also showed that let-7 is conserved throughout the animal kingdom, from humans to insects, which proved that microRNAs were a widely used regulatory mechanism, not just a fluke." In his Lasker Award speech, Ambros, who had not known about Ruvkun’s let-7 in-all-animals project, recalled being stunned by its significance: “After reading their paper in the autumn of 2000, I had to set aside 10 minutes to stare out the window and reorganize my view of the universe.” At about the same time, another RNA-based mechanism of gene regulation, RNA interference, was discovered and shown to be mediated by RNAs of the same diminutive size as the miRNAs of Ambros and Ruvkun. The biologists Andrew Fire and Craig Mello first showed that C. elegans can use double stranded RNA to silence other genes, and called the process RNA interference. In 2006 that work brought Fire and Mello a Nobel prize and brought Mello a Janssen Award. Then David Baulcombe, a plant geneticist at Sainsbury Laboratory in U.K., showed that plants generate tiny antisense-acting RNAs during RNA interference that were roughly the same size as lin-4 and let-7.

Interest in microRNAs took off. Today, a search for journal articles about microRNA turns up more than 59,000 citations—more than 52,000 of them since 2010. Cancer research is only one area in which microRNAs have risen to prominence. MicroRNAs generally seem to be less abundant in tumor cells, which suggests they may normally help to rein in the development of malignancy. The distinctive microRNA signature of individual tumor cells might therefore be useful in flagging the origins of a patient’s cancer and in identifying which courses of treatment might be most effective. MicroRNAs can circulate in the blood and other body fluids, which makes them easy targets for medical diagnostic products. The company Mirna Therapeutics is developing a cancer treatment, currently in clinical trials, based on replacing the microRNAs deficient in abnormal cells. Outside oncology, microRNAs could also one day find a wealth of clinical applications in the treatment of neurodegenerative illnesses and other conditions because they seem to influence the developmental plasticity of stem cells.

Ambros continues to be a leader in the study of microRNAs. In his laboratory at University of Massachusetts Medical School, which he started in 2008, he explores the complexities of gene regulatory networks and the role of microRNAs with them. One focus of those studies is how microRNAs help to buffer organisms against physiological and environmental stresses to improve organisms’ survival.

Ruvkun remains very active in microRNA and siRNA research. His laboratory has studied how microRNA and siRNA genes regulate their targets and has developed genetic tools to increase the effectiveness of these regulatory RNAs. Ruvkun explores other aspects of biology as well. For example, he discovered that C. elegans controls its metabolism and lifespan through a signaling pathway that depends on an insulin-like receptor molecule, just as mammals do. Beyond its practical value for suggesting new targets for future diabetes drugs, this discovery implies that the molecular pathways governing metabolism and longevity have been strongly conserved through hundreds of millions of years of evolution, a recurrent theme in his research.

Ruvkun and his colleagues are also developing techniques that could detect so-called extremophile microorganisms that might play an overlooked role in some human disease. A spinoff of that project is literally out-of-this-world: for over 20 years, Ruvkun has worked on analytical systems that future robot landers on Mars could use to search for evidence of life that shares ancestry with life on Earth. The effort is called the Search for Extraterrestrial Genomes Project (SETG). Again, Ruvkun is searching for deep conservation of biology, in this case between planets.

READINGS Gitschier J. In the Tradition of Science: An Interview with Victor Ambros. PLoS Genet. 2010; 6(3): e1000853. doi:10.1371/journal.pgen.1000853 Nair P. Profile of Gary Ruvkun. PNAS. 2011; 108 (37) 15043-15045. doi:10.1073/pnas.1111960108 © Scientific American 2017

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