How the instruction is executed

Corresponding Member of the Russian Academy of Sciences Peter Sergiev: a gene is an instruction for creating an organism

Natalia Leskova, "Scientific Russia"

Why it is necessary to understand the functions of all genes and how it can be done, whether a person will be able to create organisms with improved properties, what surprises the study of the genome opens, why look for new antibiotics and how they will fundamentally differ from the already known ones, says Pyotr Vladimirovich Sergiev, professor of the Faculty of Chemistry of Moscow State University, associate Professor of Skoltech, director of the Institute of Functional Genomics of Moscow State University, Corresponding Member of the Russian Academy of Sciences.

— Pyotr Vladimirovich, you are a hereditary scientist, your father and grandfather were academicians, epidemiologists and parasitologists. You went your own way. Why?

— I wanted to try something of my own, in my field. I really like what I do. In fact, I probably haven't hesitated in choosing a profession for a very long time since I was a child. Something between chemistry and biology — I had such a choice literally from school. As a result, I work at the chemical faculty, but I deal more with biological problems.

— Let's talk about your research on mice. What are they interesting for?

— It seems to me that now is a very romantic time in biology, something akin to the era of Great Geographical Discoveries, when more or less common features of the map, and in this case the genome, are clear, but there are still a lot of white spots to be explored. In my opinion, the most interesting thing now is to study the function of genes. There are not as many genes as it seems, there are about 20 thousand of them in almost all multicellular. But there are, of course, differences.

Many thousands of genes have not yet been characterized, the computer has only "looked" at them. It is very interesting to understand what they are programming. After all, these are the instructions with which the human or mouse body is created. It seems to me that this task is within the power of the scientific community — within the next ten years to understand the function of all genes. And then it will be the turn of engineering biology and the creation of living beings according to our ideas.

— Is it necessary to create living beings according to our ideas?

— It's inevitable.

— Why?

— There are several answers here. The first one is purely utilitarian: maybe it will be possible to create, for example, farm animals with the necessary properties.

— Or universal soldiers.

— Let's not talk about such things. We do not have such tasks, and I think this case is still fantastic and purely hypothetical.

— There really is such a plot in fantasy literature. But what was fantastic yesterday often becomes reality today.…

— Still, let's think about the good. In addition to animals with some properties we need, the creation of organisms is a fundamental knowledge, a test of our understanding. It seems to me that only by creating an organism can we assume that we have correctly understood how organisms appear and function. Until that time, no one guarantees that our knowledge will be complete.

— Let's say we have reached this level and learned how to create living beings with the specified properties. However, this is a matter of the future. What is the path to this, what are you doing now?

— We are trying to investigate the function of genes that no one knew about. There are some successes in this. For example, our narrow specialization is the study of methyltransferases that act on RNA. This is, I would say, molecular tuning. RNA is part of molecular machines, quite complex, multicomponent, such as, for example, the ribosome, which synthesizes all proteins, or the spliceosome, which cuts out unnecessary fragments in transcripts. And these molecular machines are modified, that is, chemical groups, for example methyl groups, are attached to them with the help of special enzymes. And since a special enzyme has appeared in evolution, it probably means that it is needed for something.

— Although there were points of view that we have a lot of garbage in the DNA. Then, however, it often turned out that it was still functional. Now there is no such point of view that something can be superfluous in the composition of DNA?

— There may be random garbage patches in DNA, but still, if this is a normally functioning gene and a functional enzyme is obtained when this gene is expressed, then the enzyme is certainly not accidental. For example, we have studied genes that modify the mitochondrial ribosome. Mitochondria are, as you know, the energy factories of the cell. They descended from symbiotic bacteria that were once absorbed by the archaea, and since then this archaea and the bacterium have been living together, forming a eukaryotic cell.

Mitochondria have their own protein biosynthesis apparatus, which is not completely similar to the one that most of our proteins synthesize. It looks more like bacterial. And so we found enzymes that improve the mitochondrial ribosome, participate in its assembly and modification.

We discovered two such enzymes and studied them at the cell level. They have quite an interesting and different evolutionary history. One enzyme just came from a bacterium, and this is a bacterial inheritance in our genome. We have two systems supported — one came from archaea, the other from bacteria, one modifies cytoplasmic ribosomes, the other mitochondrial, and both systems are aimed at approximately the same nucleotides.

— And the second enzyme?

— The second enzyme that we found, on the contrary, is very new, it appeared only in vertebrates as a result of the complete duplication of the genome that occurred in their evolution. The gene of the ancestral enzyme of invertebrates doubled, and one of the copies acquired a new specialization. We also created mice with knockout of one of these genes and saw a rather interesting phenomenon.

— And what does it mean — gene knockout?

— We are using the well-known and very popular CRISPR-Cas system now: we take fertilized eggs from a mouse, microinject the components of the CRISPR-Cas system that we specially create in them, and "push" them to a certain part of the genome. This system in the egg makes a break in the DNA. Such a gap can lead to damage to the gene. Accordingly, these eggs, after we have injected the CRISPR-Cas system into them, we plant the mouse, and it bears them.

This is a rather complicated and tricky operation, it takes a long time to learn. I've been learning to do it myself for a year, now I've taught the guys at Moscow State University, and they do it themselves. The mouse then gives birth to mice in due time, three weeks later.

— How do they differ from the usual ones?

— Outwardly nothing. We analyze the part of the genome that we have targeted, look to see if there have been changes there, and quite often with good efficiency we see a gene knockout. Then you can breed such mice and investigate what is wrong with them. In fact, the study of unknown gene functions so far boils down to messing up the gene and see what happens.

— What does this information about corrupted genes give?

— We see what happens to the mouse. Namely, in the case of inactivation of this enzyme, which works with the mitochondrial ribosome, we see that mice become weaker, which is logical in principle, because mitochondria provide energy to muscles. But we also saw a rather strange phenomenon — they study worse. It cannot be said that we have mouse scientists here, but we can teach them to look for a path in the maze, and it turns out that mice without this improvement of the mitochondrial ribosome learn worse, it is more difficult to remember the path in the maze, give slack in several more tests.

— Don't you feel sorry for the mice? Sorry for the unscientific question.

— It's a pity for mice, but still, it seems to me, it's worth it, because we also get knowledge about our genes. In addition to purely fundamental research, we have quite a lot of research on cooperation with medical geneticists.

— Tell us about it.

— One of them, for example, is within the framework of a grant from the Russian Academy of Sciences in cooperation with the NMIC of Oncology in St. Petersburg. We recreate mutations that medical geneticists in the field of oncology of the E.N. Imyanitov group find in patients, and this allows us to create models of predisposition to oncological diseases. In fact, medical geneticists can only assume with some degree of certainty that this or that mutation is pathological. They observe correlations in some way. But in order to establish a causal relationship, we need to recreate this mutation on a model object, for example, on a mouse, and see if we really see what we will see in patients.

— Are your knocked-out mice associated with such mutations in cancer patients?

— Yes, of course. We are actually reproducing the mutations that medical geneticists see in patients. This should help identify them at an early stage and treat them successfully.

— You have learned how to create such mutations and harm mice. And do you have any opposite experiences when, on the contrary, you improved the mouse, increased the quality of its training?

— Of course, we haven't created Pinky and Brain yet. I think nature has optimized living organisms quite well, so it's not so easy to improve them in evolutionary terms with the help of our manipulations. For now, this is a matter of the future. Although theoretically, if we take into account that horizontal gene transfer in such complex creatures is difficult, we can predict that genetic engineering will someday improve living beings.

— But we are already talking about creating plants with improved properties using gene editing techniques. You said that in the future there will be animals with optimized characteristics. That is, we are already arguing with evolution, finding a lot of flaws in it.

— It's true. By the way, we are working with the Institute of Animal Husbandry, together we are trying to create farm animals. So far, we have worked out the techniques of gene editing on animal cell lines. And our colleagues from the Institute of Animal Husbandry have worked out a methodology for obtaining animal clones by transferring from the nucleus of a somatic cell to an egg. So now we need to put these two stages together. I hope we will be able to tell something positive in the future.

— What is the uniqueness of your research?

— All scientists have their favorite areas of work where we have priority. We examined several genes that no one had thought of. For example, we have now studied the function of one of the genes, the so-called mitoregulin. This is also a gene whose product works in mitochondria, but it is not a protein, but a peptide. It was annotated by computer as a non-coding RNA gene. The computer looked at that there is a very small section that encodes a small protein in 56 amino acids. We identified it, realized that this small protein is located in the mitochondria and that it is needed for the operation of one of the complexes of the respiratory chain.

We see that without it, the composition of mitochondrial lipids changes, one of the main lipids of the mitochondria, cardiolipin, is excessively damaged there. And we observe in mice a number of pathologies associated with the work of the kidneys, with muscle strength. We actually extracted something of great value for the body from non-existence, from DNA that seemed to be garbage, and came closer to understanding how it works.

— Let's say we someday understand how all our genes work, open the genetic apparatus. Will this mean that we will know ourselves, or will there still be some riddles?

— I think that man, like all animals, is a kind of biological machine. We may not understand everything, but at least a lot. If the entire scientific community unites, it seems to me that this task will be up to him.

— Isn't it scary to interfere in the holy of holies, in the genome, in DNA, in the Creator's plan?

— We still feel like twos in this field, because, unfortunately, we often only know how to spoil.

— And this is dangerous.

— We are careful and meticulous twos. We are trying to become smarter, to understand how it all works.

— Another area of your research is related to antibiotics. Is this the creation of fundamentally new antibiotics?

— We have a whole direction related to bacterial protein biosynthesis. Probably, about half of the antibiotics are aimed at suppressing the ribosome, that is, the molecular machine that synthesizes protein. Accordingly, there is a fundamental task — understanding how antibiotics harm the ribosome. Antibiotics as tools, in fact as inhibitors, also allow us to study how the ribosome functions, how it synthesizes protein. And this interaction of the ribosome and antibiotics is fraught with quite a few more discoveries.

We have a search direction when we are looking for antibiotics both among synthetic compounds and among natural ones, which are made, for example, by soil microorganisms or other creatures. Here it is necessary to mention IA Osterman, who played a big role in this. We have come up with a system that allows us to quickly and simply determine what a particular antibiotic acts on in a bacterial cell. To do this, reporter genes of fluorescent proteins have been introduced into the cell, the expression of which depends on certain regulatory mechanisms, so that a bacterial cell, dying from an antibiotic, signals to us why it is bad.

— What does it give you?

— We can immediately understand whether a particular compound acts, for example, on protein biosynthesis or damages DNA. After that, we analyze such a compound in the in vitro system. We have established cooperation with several laboratories that are engaged in structural studies of ribosomes, and we see where the antibiotic binds. Sometimes it is possible to find some new antibiotics with a new mechanism of action. Some of these works have been published, some are still in the process of work.

But a number of antibiotics have been found — amikumacin, for example, which stops the movement of RNA along the ribosome when it reads the matrix RNA and synthesizes a protein. This antibiotic sticks mRNA to the ribosome. There is tetracenomycin, which binds in the tunnel through which the protein leaves the ribosome. This is the graduation path for protein. It sits there and blocks the path of the synthesized protein.

But here's what's interesting. If earlier it seemed that the antibiotic simply blocks a certain stage of the ribosome, now it has become clear that everything very much depends on the protein sequence that the ribosomes synthesize. It's as if we imagined that, for example, protein synthesis is a street traffic where cars move. Previously, it was believed that an antibiotic is as if the street is blocked and no one can pass, no protein can be synthesized. And now it turns out that no, it's not quite true. For example, we see that only trucks cannot pass, but passenger cars can. Accordingly, we see that different peptides are differently sensitive to the action of antibiotics.

This is a very interesting topic, it was launched at one time by A.S. Mankin, our former compatriot, who now works in Chicago, and we are also conducting these studies. Together with our colleagues from Novosibirsk (this is the group of M.R. Kabilov), we have created a new method of how to look at the specificity of antibiotics in relation to the proteins that they inhibit.

— What kind of research is this?

— We have figured out how to apply the method of high-performance sequencing. If you go into scientific details, the method is based on reverse transcription: the position of the ribosome, when it moves along the mRNA, can be determined using reverse transcription. The reverse transcriptase also moves along the RNA, if there is a primer, and, meeting the ribosome, the revertase stops. By the length of the reverse transcription product, it is possible to understand where the ribosome is stuck. This also applies to stopping the ribosome due to an antibiotic.

But this is not news, but the news is that we have learned to do it simultaneously with 40 thousand matrix RNAs and immediately get information about where the ribosome stopped, stuck because of a certain antibiotic. So we can figure out the rules.

And indeed it turns out that antibiotics act selectively, that is, they stop some proteins, some do not. Moreover, it is important for the activity of antibiotics, because if the whole process of protein biosynthesis in the cell stopped at the same time, then when the antibiotic is removed later, everything can continue. This is how a bacteriostatic antibiotic works.

— But we want to kill the bacterium once and for all.

— Yes, and it is interesting that such selectivity of the action of antibiotics is often associated with bactericidal activity, because the worst thing for a bacterium is when there is a violation of regulation, that is, some proteins are synthesized, some are not and a mess of proteins appears in the cell. Everything goes awry, and the bacterial cell dies.

— You are now talking about general patterns, but it is known that some antibiotics work in one case, but not in the other. Are these individual characteristics somehow taken into account in your research? Is it possible in principle to create a universal antibiotic that will be effective in all cases?

— While antibiotics act more or less universally on all bacteria. The problem here is that I want to create a magic bullet that would act on all bacterial pathogens. That is, it acted on pathogenic bacteria, but not on peaceful bacteria that help us a lot, for example, in digestion, in the immune system. So far, of course, this does not exist yet. There is a specificity of antibiotics, which rather interferes. Some antibiotics are strictly specific to a small group of bacteria, usually close relatives of antibiotic producers, but it cannot be said that it is pathogenic bacteria that are suppressed, and good ones are not suppressed. Let's see if we can create or find something in the future.

— Now the problem of antibiotic resistance is acute all over the world, which all doctors, especially resuscitators, are shouting about. Are you dealing with this problem? Is it possible to find such antibiotics that will not be affected in any way?

— This is a very correct question. Even just searching for new classes of antibiotics will buy time in this competition with bacteria, because this is a kind of arms race. We are looking for new antibiotics, bacteria come up with resistance mechanisms. Even a postponement in this race is already good.

However, I want to solve this problem once and for all. So far this has not been possible, but there are some pairs of antibiotics that act on the ribosome, for example, in the opposite way. This is still our secret, because the work has not been published, but it seems that we have found some kind of antipode of streptomycin.

— What is it good for?

— Streptomycin is a well—known antibiotic, discovered back in the 1940s. It affects the level of errors and the interaction of transport RNAs with the ribosome. To simplify it very much, let's say I look like a small ribosome subunit that has a head and a trunk, and here, like a scarf around my neck, there is a matrix RNA. The transport RNA comes here, the reading takes place, and then they move along the neck with a small subunit. If the ribosome understands that the transport RNA corresponding to the codon has arrived, that is, the records in the mRNA about the amino acid, the decoding center closes, that is, it presses the tRNA with its head, as it were. But only the right one, only if the tRNA came with the right amino acid — then this amino acid will be included in the protein.

— Is this what happens with streptomycin?

— No, streptomycin promotes this movement even for the wrong tRNAs. That is, he tells the ribosome that even the wrong amino acid will do, and the ribosome begins to make mistakes. This is due to a conformational change, with the movement of the ribosome head. And the antibiotic that we recently discovered works the opposite way.

— How's that? Throws his head up?

— Yes, he throws his head back, that is, the ribosome kind of lifts its nose, and this leads to the fact that the tRNA falls out. But what is most interesting is that mutations of resistance to streptomycin increase sensitivity to this new antibiotic, and vice versa. There are chances that we will catch such a pair of antibiotics that a bacterium, defending itself from one antibiotic, will inevitably expose itself to the blow of another, and vice versa.

— Will it appear in pharmacies soon? What is your forecast?

— Wait. It is necessary to understand whether it can, in principle, appear in pharmacies, whether it is safe, first of all, for a person. The biggest problem is not even to find a new effective antibiotic, but to find a safe antibiotic. It is rare to find antibiotics that can be used without risk. There are quite a few compounds that kill bacteria, but, unfortunately, they can also kill a person, so they cannot be used. So while we're on the lookout.

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