Dr. Paul Janssen Award

scientific_american

By John Rennie, Science Editor & Journalist

When the molecular biologist Craig C. Mello was a boy in Virginia in the 1960s, he was a disappointing student in the classroom. But outside, left alone to indulge his curiosity, the scientist he would eventually become sprang to life. Whenever possible he spent hours wandering through the woods, peering at leaves and animals, turning over rocks in search of wonders beneath them. His father, a paleontologist who worked for the U.S Geological Service and late the Smithsonian Institution, often brought the family with him on fossil-hunting expeditions to the American southwest, as well as on hikes and camping trips closer to home. Those trips awakened an early love of nature in Mello and a deep appreciation of how prehistoric surprises could survive into the modern world.

How fitting, then, that as a molecular biologist Mello ushered in a revolution in biomedical research with the discovery of an ancient, long-overlooked mechanism that cells use to control gene expression. He and its co-discoverer, Andrew Z. Fire, dubbed it RNA interference, or RNAi, and it appears to be centrally important for enabling organisms to protect themselves from viruses, to adapt flexibly to changing environments, and to maintain stable, well-differentiated tissues. So significant was their finding that in 2006—just eight years after its publication—they were jointly rewarded with the Nobel Prize for Physiology or Medicine, and Mello received the inaugural Dr. Paul Janssen Award for Biomedical Research.

Biological mechanisms are far more stable than the positions of continents and oceans on the face of the earth, and could even outlive the earth if we find a way to colonize space…"As Mello wrote in the autobiography he provided to the Nobel committee, "The biological mechanisms at work inside our cells are truly ancient and remarkably stable, more stable even than the positions of continents and oceans on the face of the Earth."

Mello's boyhood belief that he would become a scientist became irrevocable in the late 1970s when he heard the news that scientists had cloned the human gene for the hormone insulin into bacteria, making it possible to produce limitless supplies of what had been an expensive therapeutic compound. Fascinated with the promise of molecular biology, Mello enrolled at Brown University to study chemistry, which he followed up with further doctoral work at the University of Colorado and Harvard University.

It was during his graduate studies that Mello focused his attention on the nematode Caenorhabdytis elegans. A tiny, transparent soil roundworm with neither a respiratory nor a circulatory system, C. elegans is only a millimeter long and about 1,000 cells in size. Primitive though it might seem, it is exquisitely well-suited to studies of animal development. Investigators have been able to document how every one of those 1,000 cells differentiates and takes up its location in the adult animal. (In 1998 C. elegans also became the first multicellular animal to have its entire genome sequenced.)

During his graduate studies, Mello began to collaborate with Andrew Fire at the Carnegie Institute for Science in Baltimore on a way to insert genetic information—a technique called DNA transformation—into C. elegans. They eventually succeeded and that discovery became the basis for Mello's doctoral thesis in 1990. Mello then moved to the Fred Hutchinson Cancer Research Center in Seattle to continue his studies of the genetic regulation of nematode development. In 1994 he moved to the University of Massachusetts Medical School.

Mello, Fire, and their colleagues experimented with controlling genetic expression in C. elegans by injecting the animals with single strands of so-called antisense RNA—sequences of ribonucleic acids that were complementary to the messenger RNA (mRNA) molecules transcribed from active genes. In effect, antisense RNA turned off genes by binding to their mRNA and prevented it from being translated into proteins.

But Mello, Fire, and other researchers working with C. elegans also noticed that injecting single strands of sense RNA—ones with sequences identical rather than complementary to mRNA molecules—could also selectively shut off genes, which was highly unexpected. And a further, bigger surprise awaited Mello and Fire when they injected paired strands of RNA into the roundworms: the double-stranded RNA was vastly more potent at censoring a matching gene than the single strands were. No more than a few molecules of paired RNAs per cell were needed to obliterate a gene's expression. Moreover, the suppression of the gene could sometimes persist into subsequent generations of the roundworms. These observations dovetailed with peculiar results that other scientists had reported previously while experimenting on petunias and tobacco plants with antisense RNA and multiple copies of genes.

Mello and Fire realized that a novel gene-expression mechanism involving RNA could explain what was happening. They called it RNA interference and described it in a groundbreaking paper in 1998.

What they discovered was that when paired strands of RNA 20 to 30 nucleotides long are injected into a cell, a series of reactions peels them apart and links them to specialized enzymes called argonautes (or AGOs). The argonautes use these RNA templates to check for complementary mRNAs in a cell millions of times per second. When an argonaute finds close matches, it can destroy those mRNAs or enlist other proteins to block their translation, thus interfering with a gene's expression. This mechanism is so powerful and efficient at locating targeted RNAs that researchers in the field frequently liken it to a genetic "search engine."

RNAi appears to have evolved at least a billion years ago. It may have first arisen to help subdue viral infections, because the double-stranded RNA construct bears similarities to bits of viral genome. But whatever its original function, RNAi now operates in cells as part of a regulatory network that silences specific genes in ways that go beyond the traditional controls on transcribing and translating DNA. Indeed what makes the idea of RNAi so revolutionary is that it shows that the transcription of a gene is not just for the purpose of expressing a gene: transcription can be part of a feedback loop for regulating the gene, too.

One advantage of RNAi for cells is that it hugely increases the efficiency with which they can fine-tune the expression of their genes. For example, if a cell's environment abruptly changes, the cell may be stuck with leftover, untranslated mRNAs that in theory would commit it to a response that is no longer useful. RNAi can purge the cell of those obsolete mRNAs almost instantly and hasten the cell's adaptation to its current situation.

The more astonishing effect, however, is that RNAi's regulatory effects can pass from one generation to the next: offspring can inherit some of their parents' adaptations to an environment without a genetic change. This phenomenon of epigenetic inheritance has recently become the object of intense biomedical interest as researchers try to understand how passing environmental conditions may sometimes alter the physiology of people and other organisms throughout their lives and into later generations.

Now that the potency of RNAi at regulating genetic expression is known, the challenge is to turn it into a useful tool of biotechnology. As an experimental tool, it has already proved itself countless times in studies of basic gene function. But adapting it as a type of therapeutic drug compound, for instance, is complicated: because RNAi molecules are large and polarized, they do not easily or efficiently pass through the lipid membranes around cells, which prevents ones administered artificially from having their desired effects.

Mello has interests in developing RNAi applications, but much of his own research focus at present is on studying how RNAi and a menagerie of single-strand, non-coding RNA species naturally regulate genetic expression. (Much of his current work centers on tiny single-strand snippets called piwi RNA.) And as in the earlier stages of his work, C. elegans remains an indispensible partner as the research subject of choice. Studies in 2012 by Mello's group, for example, showed that the reproductive cells of C. elegans use complexes of short single-strand RNAs and argonaute proteins to scan their genome and silence the expression of parasitic sequences from viruses.

Mello moved from roundworms to gypsy moths for a 2014 study of the importance of RNAi to the insect's immunological defenses. Normally, certain viruses that can infect a wide variety of insect hosts have little success in attacking gypsy moths. Mello and his colleagues learned that the moths' resistance springs from natural RNAi molecules in their cells that block the replication of the viruses. But then the researchers also artificially infected the moth cells with vaccinia, the virus best known for its role in the development of a smallpox vaccine. Vaccinia disrupted the moths' protection against the other viruses, possibly by interfering with the RNAi mechanism's ability to target viral proteins for destruction.

That spirit of discovery that motivated Mello to wander through forests and dig for fossils when he was a boy has never faded. For him, the goal of understanding nature remains the noblest pursuit conceivable. As he noted in his Nobel autobiography, "The world is a far more remarkable place than we can imagine. Its mysteries define the human condition; to exist without knowing why." And as for himself? "I haven't changed much really in the intervening years. I'm still turning over stones, hoping to find something new."