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Constructive interference

 

Olivier Voinnet is Professor and Chair of RNA Biology at the Swiss Federal Institute of Technology Zürich as well as Directeur de recherche Détaché du Centre National de la Recherche Scientifique (CNRS), France. In 2009, he was awarded the EMBO Gold Medal for his work on RNA silencing in plants. In EMBOencounters, he talks to Barry Whyte about his career and recent papers in Nature Genetics and Science.

 

How did you first get involved in gene silencing research?

Before my position at the CNRS in 2002, I worked with David Baulcombe, who is now Professor of Botany at the Department of Plant Sciences at the University of Cambridge. When I joined David’s lab in 1996, very little was known about gene silencing; some people even thought it was an artefact of transgenesis. But we managed to develop a model system that helped work out some mechanisms and uncover a universal role for RNA silencing in antiviral defence. Our recent findings in mammals make me more inclined to believe that defence against viruses and transposons constitutes the primordial function of RNA silencing across all kingdoms of life.

 

This was before we knew the identity of small interfering RNAs or microRNAs?

Yes. Small interfering RNAs or siRNAs were discovered in David’s group at the Sainsbury Laboratory in Norwich, England, in 1998, right in the middle of my Ph.D. These are the infamous small double-stranded RNA molecules, typically 20-25 base pairs in length, that provide sequence specificity to the whole mechanism of RNA interference. We rapidly realized that virus-derived siRNAs accumulate in virus-infected plants and that these molecules underlie the specificity of the antiviral immune system we had just discovered. I have fond memories of this time and particularly remember the perseverance of Dr. Andrew Hamilton, the postdoctoral researcher in charge of the siRNA project. He was absolutely convinced that antisense molecules had to be involved in the process and he considered all possibilities to eventually find them, even though they were such tiny molecules. This tenacious and uncompromising attitude remains one of my greatest inspirations in science, alongside the incredible support and freedom I received from David to conduct my research at an early stage. I was very fortunate to be there at the right time with the right tools.

 

It was really in the early 2000s that microRNAs were recognized as a distinct and large class of biological regulators, even though the first micro- RNA and its target had been reported many years before in Caenorhabditis elegans, thanks to the seminal work of Victor Ambros and, later, Gary Ruvkun. microRNAs are similar in size to small interfering RNAs and processed by the same enzyme, Dicer. They are encoded by endogenous genes and regulate cellular gene expression. They were found in many organisms, including plants, although for some reason the plant community was a bit slow to hop onto the “microRNA train.” Small RNAs were breakthrough of the year in Science in 2002, but, from my perspective, the siRNA and miRNA breakthroughs had already been made many years before: there is a big difference between making the key discovery and generalizing a discovery. There are some interesting historical perspectives to be made about this field in this respect.

 

How important has work in plants been to the understanding of how RNA interference works in mammals?

The discovery of siRNAs in David’s lab is certainly, in my mind, the most important contribution of the plant work to the whole field. For the first time, readily detectable molecules, the siRNAs, could be used to ascertain, initially at least qualitatively, that RNA silencing was at work in a tissue or under specific conditions. Before siRNAs were discovered, all we could do was measure the loss of mRNAs, which is not very useful to characterize a process. Using siRNAs as molecular markers expedited the elucidation of the basic mechanism of RNA silencing.This was convincingly done in animal model systems, mainly Drosophila. The same basic mechanism was found to function in mammalian cells where it was and continues to be studied from a gene regulatory standpoint in the framework of miRNA-mediated regulation.

 

Other key concepts that emerged from our plant work included the notion that many plant viruses encode suppressors of RNA silencing, as is most likely the case for most invertebrate viruses. These viral suppressors form a large array of proteins that have proved to be very useful tools to dissect the silencing mechanism itself. We and others also found, very early on, that RNA silencing moves between cells and over long distances in plants and we showed how this process forms the systemic arm of antiviral silencing, emphasizing further its analogy to an immune system. But the plant contribution to the RNA silencing field went well beyond antiviral defence. In particular, the phenomenon of small RNA-directed DNA methylation and chromatin modification was discovered and extensively characterized in plants, and this created a whole field of investigation with important ramifications in fission yeast, flies and even, possibly, mammals. Amplification of RNA silencing was also discovered and characterized in plants. Many people do not realize that Dicer was first identified in a plant genetic screen. Even the name ‘ARGONAUTE’, which defines a universal class of RNA silencing effector proteins, stems from the squid-like phenotype of an Arabidopsis mutant.

 

In 2002, you set up your first laboratory in Strasbourg, France. What was the biggest challenge in building up your research group?

I do not recall many big challenges but you tend to forget the difficulties when you are hungry to test your hypotheses and explore new ideas. I chose the Institut de Biologie Moléculaire des Plantes in Strasbourg because I knew the infrastructure was there to conduct the type of research I had in mind. I also benefited from a CNRS incentive, called ATIP, which provides some financial support to young principal investigators. I was very lucky to attract talented postdoctoral researchers and students so that the laboratory did very well from an early stage. We received many awards and scientific prizes and this created a foundation for further discoveries. The Agence National de la Recherche (ANR) was also created at the time, and for many French scientists this was a god-send: for the first time, French research was provided with a credible, competitive funding scheme. Particularly important were the open-calls, the “ANR Blanc,” which allowed funding of basic science. The ERC system was also created at this time and our team benefited from the first competitive grants awarded in 2008. This was an incredibly productive time where we grew to a team of more than 25 researchers and expanded our research into mammalian systems.

 

And then you moved to Switzerland?

Yes. I had exhausted all credible and legal possibilities to maintain a group of this size in France when a clear opportunity presented itself at the Eidgenössische Technische Hochschule Zürich (ETH Zurich). I asked the CNRS for a “dètachement” using a unique prerogative of permanently employed French researchers. My move coincided with the decline of the ANR, where bureaucracy, lobbying and an increasing emphasis on applied science had progressively supplanted the original, highly praised ambitions of this agency. I suppose the financial crisis that hit France and so many countries did not help, but there are still inexplicable decisions that were taken about the function and scope of funding for the ANR. Given the more recent constraints imposed on the employment of research staff in state-funded institutions, I think the current conditions deter even the most motivated of French researchers, something I witness directly in our struggle to maintain a laboratory in Strasbourg. But there is still hope: France hosts some of the brightest and innovative minds and I never cease to be amazed of how much is achieved with so little. 

 

The level of support and trust given by Switzerland to its researchers is staggering and notable at all stages of a scientific career. The system is highly competitive and entirely based on merit, something I also see within the ETH Zurich itself. The strong support from Swiss funding institutions is also intertwined with a clear vision that exploratory and applied sciences do reinforce one another, and that there is space for both.

 

What has your recent work focused on?

We have many interesting projects brewing in the lab, which reflect the diversity of talented people who contribute to my laboratory. Surprisingly, defensive roles for RNA silencing have been somewhat overlooked in mammals over the years. I say surprisingly because our initial findings on plant antiviral silencing were later corroborated almost identically in invertebrates, including insects, and, more recently, in C.elegans; whether, likewise, defensive RNA silencing persisted in mammals is a legitimate question to ask. We have approached this question from two major angles that we had already taken in plants: antiviral defence and maintenance of genome integrity through transposon taming. Unlike most other researchers working on differentiated cells, I became convinced that multipotent or progenitor cells, which have now been characterized in many tissues of developing and adult mammals, would be primary sites of defensive RNAi. We now have papers in Science (1) and PLoS Genetics (2) that strongly support this idea for defence against viruses and transposons. 

 

When I moved to Zürich we had just demonstrated that thousands of cellular small RNAs are transported from cell to cell and over long distances in healthy plants. We proposed at the time that this mobile component to endogenous RNA silencing could help plants to adapt their new growth and perhaps their progenies to environmental cues or stresses perceived very locally in single leaves or roots; we now have experimental evidence to support this view and have also isolated what I believe are the first components of the mechanism that physically transports small RNAs between plant cells. Intriguing observations suggest that non-cell autonomous silencing might also operate in mammals, a question my group is now pursuing as part of a major research project on extracellular RNAs that has just been funded by the National Institutes of Health in the United States (3).

 

And now you have papers in Nature Genetics and Science?

The paper in Nature Genetics (4) which was published a month ago, describes how we were able to reactivate and follow, for the first time, the whole mechanism by which Arabidopsis eventually silences an invasive retro-element with the potential to amplify itself many times in a genome. Essentially, we have deciphered the series of molecular events that lead to the silencing of the many copies that originated initially from a single-copy transposon. We could also evaluate the significant contribution of the mobilization of this transposon to the genetic diversification of its host before the transposable element became silenced permanently. This is a heavyduty piece of work, full of genetic tricks and elaborate molecular biology experiments. We took the time to design our experiments very meticulously and we set the bar very high to decipher the whole thing from A to Z. I know our community appreciates this paper and I am proud of what was achieved here, sometimes against all the odds. I keep telling people in the laboratory that perseverance is the essence of innovative science. An obvious corollary is that exploratory science needs time and unconditional support, which brings us back to funding.

 

And the Science paper describes work in mammals?

Yes and it is yet another example of perseverance. I initiated this project many years ago, at a time when some colleagues were puzzled about my new interest in mouse embryonic stem cells (ES cells). Thanks to a very fruitful collaboration with Edith Heard at the Institut Curie in Paris, we set up the ES cell system in our laboratory, and together made some interesting observations potentially linking X chromosome inactivation and transposable elements. These findings are at the origin of our current interest in how RNA silencing contributes to silence transposons in ES, and, more widely, in multipotent cells. But Edith knew from the beginning that my real motivation to work with ES cells was to implement an experimental virus infection system because I was convinced that ES cells would be the right material to demonstrate antiviral RNAi in mammals. Mouse ES cells are also unique because, unlike differentiated cells, they can withstand the complete removal of the RNA silencing machinery, which was for me essential to conduct clear-cut genetic experiments as we had done previously in plants to decipher the molecular underpinnings of antiviral silencing.

 

The role for RNA silencing in mammalian defence against viruses has been hotly contested mostly because long double-stranded RNA produced by viruses, the molecule that initiates antiviral RNA silencing in plants and invertebrates, activates innate immune pathways unique to vertebrates. These pathways promote the non-specific antiviral interferon response, which most people consider so potent that it probably rendered RNA silencing superfluous in mammals. This argument was fuelled by the negative results of several labs exploring the accumulation of virus-derived siRNAs in virus- infected mammalian cells. We provide in the Science paper several key reasons why antiviral RNAi has remained elusive so far in mammalian cells. One reason pertains to the differentiated versus multipotent state of cells. Multipotency seems to correlate with poor interferon signalling and a tolerance to long double-stranded RNA accumulation. The second reason pertains to the use of highly virulent viruses, which, as shown previously by others and our group effectively suppress RNA silencing in plants and invertebrates. It seems obvious that you cannot use a tool to probe a phenomenon that is coincidently suppressed by the very same tool. We were not deterred by the negative results of others and persevered with our original idea, applying the knowledge we gained from our studies of plant model systems.

 

We have published in Science at the same time as a group led by my colleague and friend Shou-Wei Ding who has shown similar findings using different approaches. A staggering aspect of their work is to show that virus-derived siRNAs that immunize mice in vivo are identical in their distribution, biochemical properties and relative proportions to the siRNAs we detect in ES cells. This implies the existence of a previously unknown siRNA-based immune system in mammals. These are real breakthrough findings, and I cannot wait to get the results of ongoing experiments conducted in my and Shou Wei’s laboratories to uncover how, when and where this immune system operates in mice. I think it is really an exciting time to be working in this research area and we have still only scratched the surface in terms of biology and, perhaps, applications.

 

 

 

 

Olivier Voinnet

Olivier Voinnet

Professor and Chair of RNA Biology at ETH, Zürich
Directeur de recherche Détaché du CNRS, France
EMBO Member since 2007