“RNA should in a way be an icon of our culture.”
Juan Valcárcel tells us about his career, alternative splicing research including its relation to cancer and his view on research networks.
What made you become a scientist?
As a child, I was very curious about the nature and composition of things. When I was twelve years old, I got interested in chemistry and with thirteen I learned about the genetic material. This convinced me that I wanted to understand how DNA ended up producing living organisms.
Do you have scientific role models?
Charles Darwin for example. As a child, Fleming looked to me as the ultimate scientist because he looked like a normal human being, forgetting about plates for months and not keeping the most pristine bench. What he made out of a simple observation, which initiated a whole field, was amazing. For me, Seymour Benzer was also someone that I admired because he showed how by just doing very simple experiments with bacteriophages he was able to infer the nature and structure of the genetic material.
Can you tell us more about Fleming's discovery of antibiotics?
I have become very interested in this story and I believe that I have read every book that has been published about this and recently also visited the Fleming lab museum in London. There were two schools at the time: one wanting to help the organism to fight infections and the other advocating the use of chemicals. Fleming was working in the group of Sir Almroth Wright, who belonged to the school promoting the natural defense. Fleming's first main discovery was the enzyme lysozyme, which fights microbes in a natural way. The observation of the antibiotic phenomenon was like treason to his school. Nevertheless, he followed up on this observation, because this could bring essential advances to fighting infectious diseases, which was his research focus. There are many interesting sidewalks to this story, such that he named the compound but never isolated it. The compound's isolation was done more than ten years later by chemists in Oxford.
Coming back to Darwin, can you comment on Craig Venter's reenactment of Darwin's voyage with the Beagle applying genomics techniques?
This is great and has produced a lot of interesting information about the genomics of ecosystems. There is a nice metaphor because, in fact, Venter is now looking for weird organisms traveling the world. But I would argue that today's equivalent attitude of the naturalists in the 19th century of going into expeditions to find other organisms and how life looks like elsewhere, is looking via the computer into the databases. In these, you have the sequences of thousands of species. So for me the naturalist today is the person who sits in front of the screen and looks into the huge vast unknown that is behind these sequences. If you are smart enough to come up with exploration tools you can make discoveries that are tremendous without getting out of your living room. This allows exploration of the natural world.
Given the importance of high throughput methods and the need for tools to analyze these large datasets, would you decide today to first obtain a degree in informatics or statistics before going into life sciences?
I do not know if this would be first, but this is an absolute need not only if you are working in science but almost for anything. I have seen that these tools are as essential as any basic experimental tool. This is a crucial aspect to take into consideration when thinking about training our students. From the first year of university they should be able to be literate in computational analysis, especially bioinformatics. Without that, you are totally handicapped not only regarding the basic understanding of programs and operations that you can do in terms of computational biology, but also regarding your mind frame. Our textbooks are full of pathway schemes such as the flow of metabolites that look like linear pathways with some cycles present. Today it is apparent that we have to look at the unity of the genome and the organism. The processes are talking to each other in fascinating ways. These networks of interactions between components at any level are essential. When we do a knockdown of a gene, what we are in fact doing is perturbing many other genes, and this is a nuisance if you want because then these linear pathways are no longer going to be there. However this is reflecting a very profound reality of how living organisms are built. Everything they do is based on these networks of interactions at the gene, protein, organelle level and also between the cells forming an organism. We have to be able to understand systems from a systems perspective, and without that, we are going to be very limited.
Could you share with us a defining moment in your career?
My PhD thesis project was supposed to be a search for genes in the influenza genome that are important for the generation of variability. This was a fascinating topic for me, but I was doing this side project trying to express a gene from the virus, which is alternatively spliced. When I did that expression through the genome of another virus, I realized that the pattern of alternative splicing was changed entirely. This was for me the first time that I had discovered something that was not in a textbook and nobody had seen before. I then started to look into the process of alternative splicing and learned that there was almost nothing known about it. What was known at the time was that alternative splicing was happening in different tissues in different ways. There was such a disconnect between the very little that was known and the perspectives that were opened by the possibility of modifying the readout of genes. I thought it could be fantastic to learn how splicing is regulated and I was very keen to move into this field. This was the most defining moment in my whole career, because I am still obsessed researching the splicing machinery and how it works.
You have been working in the splicing field for over three decades: What future developments do you expect given the developments in the last decades?
So to be honest, I feel pretty much the same that I was feeling that day when I realized that there was so little known about splicing and that there was so much to explore. We have nowadays much better technologies, knowledge and have identified the components of the machinery which we think are part of it. However, we are still almost unable to predict how tissue-specific splicing is established and are lacking even basic concepts about how this works. Even for the best studied alternative splicing factors, such as hnRNP proteins or SR proteins, we only have a basic understanding of their mode of action. The process of exon definition is not understood at all. Noncoding RNAs could be involved in this process, and I dream of a time when maybe there will be noncoding RNAs that will bring the splice sites together by base pairing bringing a simple explanation for alternative splicing. I know that most likely this idea is wrong, but we have not ruled out that such a mechanism exists. Also, the coupling of splicing with other gene expression processes is only starting to be understood. The latest data suggest that splicing can take place a few nucleotides after the RNA gets out of the polymerase. So the two machineries are in very close contact, and we do not understand their interactions. Then the possibility to modulate the process through understanding its mechanisms would open up an entirely new way to ask about the functions of genes or to correct the malfunction of genes, such as the recent developments with antisense oligonucleotides or small molecules to modulate splicing.
“If you are smart enough to come up with exploration tools, you can make discoveries that are tremendous without getting out of your living room."
Your group researches the splicing of exon 6 of the FAS gene. Would it be enough to kill a cancer cell by switching the splicing outcome to the pro-apoptotic protein isoform?
Well in some context it may contribute. The gene that I think is most involved in killing cancer cells, which is alternatively spliced, to yield pro- and anti-apoptotic isoforms, is Mcl-1. When we look at alternative splicing changes after treatment with a cytostatic drug, for most such drugs the most affected alternative splicing event is in Mcl-1. So I think this is a critical one to study. There are several others like Bcl-x and FAS. Overall, there might be a program of apoptosis mediated alternative splicing that could be exploited for treatment. Another fundamental question that is not solved is why cancer cells are more susceptible to splicing modifying drugs? Why do they change their splicing much more in response to these drugs than other cells? Is it a matter of membrane permeability or how their splicing machinery is affected? Related to that, there is a very interesting concept called synthetic lethality. It appears that very often cancer cells change their alternative splicing because of for example accumulation of mutations in the splicing machinery components. This gives a cancer cell a particular advantage. For example, mutations in SF3B1, which is one of the core components of the machinery, activate the use of cryptic 3' splice sites located a bit upstream of the canonical 3' splice sites. It has been shown that these splicing changes advance tumor progression. But at the same time, this same mutation causes quite a lot of trouble because of other alterations in splicing accompanying this mutation. This makes these cells particularly sensitive to splicing inhibiting drugs. So you have a sort of synthetic lethality of this mutation with splicing inhibitory drugs, which cause in normal cells a certain amount of disarray, but much less than in cancer cells. So, what has provided an advantage is at the same time sort of an Achilles' heel for the cancer cell. This phenomenon was observed in several different types of cancer. Another interesting case is in melanoma: Initially, melanoma can be treated quite effectively with drugs like Vemurafenib, which is a B-Raf inhibitor. The problem is that after some time the tumors become resistant and in some cases, it has been shown that there occurs an activation of a cryptic splice site in B-Raf that removes the region of interaction with the drug. The tumor cells had to relax its splicing in a way to generate this variant, which is perfectly good for the tumor to progress, but at the same time, this makes the cell more sensitive to splicing inhibiting drugs. This is another example of the concept of synthetic lethality. There are clinical trials underway with drugs that should be particularly effective when there are mutations in spliceosome components present.
Do you have interactions with clinicians?
Not for the drug-related projects, which are still pre-clinical, but we do for using alternative splicing as a diagnostic marker. In breast cancer studies, which we have done together with the Hospital del Mar in Barcelona and the Institute Curie in Paris, we could correlate the response to chemotherapy with the particular isoform ratios in specific oncogenes.
What is your general view on translational research?
This is a very important aspect of research, which has to be cultured and nurtured, but this does not mean that all research should be translational. This would make no sense at all, but there is no harm in following up the translation angle of a basic research finding whenever possible. The majority of the leads from basic research are never going to result in a translational application, therefore the more leads one explores, the better.
What do you consider the most significant recent findings in the field of RNA Biology?
I think that almost everybody would agree that the potential of the CRISPR system to edit the genome was a finding that has given spectacular results and has amazing potential. Closer to the field of splicing were the therapeutic effects found in clinical trials with antisense oligonucleotides to treat Spinal Muscular Atrophy. This is fantastic news for science in general and for the field in particular, because it shows that modulation of RNA metabolism has therapeutic value, which was long believed to be an utopia. We will probably see much more of this in the future, and maybe in 10 years, the picture is going to be very different regarding therapies and applications. This is very exciting!
Regarding long noncoding RNAs, do you expect significant progress in identifying their functions or rather that it will be found that most of them are just byproducts of the transcription of nearly the entire genome?
I think that likely there will be many important discoveries made in this area. Regarding the percentage of these transcripts which have a functional role, the jury is out and is going to be out for a while. To me, it is entirely unclear at the moment what that fraction could be. One has to remember that even if the transcript itself has no function, the fact that it is being produced could have a role in transcriptional regulation. It is important to be very rigorous when assigning functions: correlations are good but just as a starting point. But proving function requires deletion and rescue experiments in simple systems that allow causality to be established.
“What has provided an advantage is at the same time sort of an Achilles’ heel for the cancer cell."
What is your opinion on funding large collaborative project grants potentially at the cost of less individual PI grants?
I hate the part of "at the cost of" because both are so necessary. When I was postdoc, there was a big debate about the human genome project. There were arguments that this is going to take so much money away from basic research projects that yield mechanistic understanding that this would drag the field of biomedical research for many years. They argued that this is a disproportionate investment to be made. However who today would doubt that this was a worthwhile effort? Not only because of the outcome but also because of the technology that was developed together with that project. Today, we can sequence a genome in one afternoon at the price one million times lower than the first genome. But shall we replace all the smaller and mechanistic projects by these large projects? Of course not, because otherwise we empty the basis for understanding. One needs to have both.
Can you comment on the role of collaborative grants such as the European Alternative Splicing Network (EURASNET)?
They have a very important role as well. EURASNET really helped to integrate researchers in the field of alternative splicing all over Europe. It was especially important for young scientists starting their labs to be surrounded by a nurturing environment that would provide them with opportunities for collaboration, in ways that would not have been possible otherwise. The typical example is someone that would come for example with a new technology and that would all of a sudden start to establish five different collaborations out of a first meeting with the EURASNET consortium, which would then lead to excellent publications and sometimes long-term fruitful collaborations. It was really a very important way to integrate people to ensure that they could apply complementary expertise and approaches. From people that were very interested in structural biology to people interested in medical genomics, and this allowed to bridge from the structure of a specific protein-protein interaction to patients. This was something that would have been otherwise extremely difficult to achieve. The EURASNET members were also very involved in trying to streamline procedures for example for transcriptomics. At the time, there was a big debate about microarrays, different ways of analyzing their data, RNA sequencing for transcriptomics and the transition between the two. What the consortium did was to do pilot experiments to compare results and then we could offer the members of the consortium a streamlined platform to analyze their data. This joint effort, which produced something of general value for the community would have been challenging to achieve otherwise.
What effects of EURASNET continue to be there after it ran out?
EURASNET, in the end, got almost 12 million Euros funding from the EU, which allowed us to integrate the field, and after it should have continued by itself and it has continued. For example we keep having meetings roughly every year and a half without EURASNET support and still the majority of former members of the consortium keep coming regularly to these meetings, because we find it is important to talk to colleagues and get to know about the latest developments in a friendly and open atmosphere. And also to maintain and start new collaborations. So this is a so far sustained effect of EURASNET, and hopefully, this will continue. I would ask the EU to reconsider to maintain, if not at the same level, some funding after the grant has ended, especially for the young people starting their groups. If they could have some extra funds through this scheme, then this could push them to be able to really do more and to integrate themselves into this network. This would have a really important impact and would not require a massive amount of funding for them to explore things together with the consortium. Every two to three years, there could be a new generation of young people entering the network. Pushing ahead the careers of young group leaders was the most important outcome of EURASNET, and it is a pity that this is lost.
What impression do you have so far of the NCCR RNA & Disease and from your experi-ence as deputy-director of EURASNET, what advice would you give to the NCCR?
Besides the meetings in the context of my visit, I know a little bit about the NCCR because my wife Fátima Gebauer is a member of its review panel. I have a lot of admiration for what you have done, and this is already having a great impact in Switzerland and is going to be great, especially for the young people. My advice would be, even if you have potentially over eight years to go, to start thinking of ways in which you can either lobby or establish structures that will keep the interest afterward. In Switzerland, you might be in an especially good position to follow this up by trying to convince the industry that there are important opportunities in RNA research. Such that the industry is aware of this and there might be jobs there but also possibilities for collaborations. Related to this, I would provide opportunities and training to young scientists, who would like to startup companies or engage in innovative activities.
What role does the RNA society, of which you currently serve as its president, and its annual meeting play for the field?
This was established many years ago by Tom Cech and Olke Uhlenbeck using a surplus of money they had in the bank from a meeting they had organized, and this was sort of illegal to have. So they used this money to start the RNA Society, which was a group of friends that were thinking about how to push RNA research forward. I think that this still is the spirit of this community and there are huge opportunities from the scientific point and biotech point of view. It provides a forum for people; they can meet and talk about RNA research all the way from non-coding RNAs in bacteria to oligonucleotides that will be important for the treatment of neurodegenerative disease. This forum serves to exchange information and technologies. The Society would like to serve the community of RNA researchers as much as possible, and I hope that we can find new ways and we are always open to suggestions from members. I am currently trying to push to have a more organized mentoring system through which the young scientists can rely on experienced researchers with many years of background in the field to give them sound advice regarding the next step in their career and how they think the field will evolve. Another critical aspect is to spread the word about the beauties and opportunities of RNA research to other communities, especially the medical, biotech and pharmaceutical communities, as well as to the public. Most educated citizens know what DNA is but may be not what RNA is. Despite the fact that RNA is as important, if not even more, more interesting and more versatile than DNA. However, this has not permeated popular culture. RNA should in a way be an icon of our culture.
Biography Juan Valcárcel
Juan Valcárcel obtained his PhD in 1990 from the Universidad Autónoma de Madrid under the supervision of Juan Ortín, during which he studied how the processing of the influenza virus pre-mRNA is regulated. For his postdoc, he moved to University of Massachusetts Medical Center to the lab of Michael R. Green, where he continued to work on splicing, researching the mode of action of U2AF65 and splicing regulation in Drosophila. From 1996 to 2002, he was a group leader in the Gene Expression Research Unit at the EMBL Heidelberg. Afterwards, he became a senior scientist at the Centre for Genomic Regulation in Barcelona and Professor at the Institució Catalana de Recerca i Estudis Avançats (ICREA). He served as the deputy coordinator of the European Alternative Splicing Network (EURASNET) and is currently the president of the RNA Society. In 2004 Juan Valcarcel was elected EMBO member and in 2014 awarded an Advanced ERC Grant for studying "Mechanisms of alternative pre-mRNA splicing regulation in cancer and pluripotent cells (MASCP)"
Interview by Dominik Theler