Gene expression and small RNAs in oncology. See what "Small RNA" is in other dictionaries Micro-RNA in Oncology

Scientists believe that incorrect expression of small RNAs is one of the causes of a number of diseases that seriously affect the health of many people around the world. Among such diseases are cardiovascular 23 and oncological 24 . As for the latter, this is not surprising: cancer indicates anomalies in the development of cells and in their fate, and small RNAs play a crucial role in the corresponding processes. Here is one of the most revealing examples of the huge impact that small RNAs have on the body in cancer. We are talking about a malignant tumor, which is characterized by incorrect expression of those genes that act during the initial development of the organism, and not in the postnatal period. This is a type of childhood brain tumor that usually appears before the age of two. Alas, this is a very aggressive form of cancer, and the prognosis here is unfavorable even with intensive treatment. The oncological process develops as a result of improper redistribution of genetic material in brain cells. A promoter that normally causes strong expression of one of the protein-coding genes undergoes recombination with a specific cluster of small RNAs. Then, this entire rearranged region is amplified: in other words, many copies of it are created in the genome. Consequently, small RNAs located "downstream" than the relocated promoter are expressed much more than they should. The level of content of active small RNAs is approximately 150-1000 times higher than the norm.

Rice. 18.3. Alcohol-activated small RNAs can bind to messenger RNAs that do not affect the body's resistance to alcohol. But these small RNAs do not bind to the messenger RNA molecules that promote such resistance. This leads to a relative predominance of the proportion of messenger RNA molecules encoding protein variations associated with alcohol resistance.

This cluster encodes over 40 different small RNAs. Actually, this is generally the largest of such clusters that primates have. It is usually expressed only at an early stage of human development, in the first 8 weeks of embryonic life. Its strong activation in the infant brain leads to a catastrophic effect on genetic expression. One consequence is the expression of an epigenetic protein that adds modifications to DNA. This leads to wide-ranging changes in the entire DNA methylation pattern, and hence to the abnormal expression of all sorts of genes, many of which should only be expressed when immature brain cells divide during the early stages of an organism's development. This is how the cancer program is launched in the baby's cells 25 .

Similar communication between small RNAs and the cell's epigenetic hardware can have a significant impact on other situations when cells develop a predisposition to cancer. This mechanism probably leads to the fact that the effect of disruption of small RNA expression is enhanced by changing epigenetic modifications that are transmitted to daughter cells from the mother. In this way, a scheme of potentially dangerous changes in the nature of gene expression can be formed.

So far, scientists have not figured out all the stages of the interaction of small RNAs with epigenetic processes, but some hints about the features of what is happening still manage to be obtained. For example, it turned out that a certain class of small RNAs that increase the aggressiveness of breast cancer targets certain enzymes in messenger RNAs that remove key epigenetic modifications. This changes the pattern of epigenetic modifications in the cancer cell and further disrupts genetic expression 26 .

Many forms of cancer are difficult to track in a patient. Oncological processes can take place in hard-to-reach places, which complicates the sampling procedure. In such cases, it is not easy for the doctor to monitor the development of the cancer process and the response to treatment. Often, physicians are forced to rely on indirect measurements - say, on a tomographic scan of a tumor. Some researchers believe that small RNA molecules could help create a new technique for monitoring the development of a tumor, which also makes it possible to study its origin. When cancer cells die, small RNAs leave the cell when it ruptures. These small junk molecules often form complexes with cellular proteins or wrap themselves in fragments of cell membranes. Because of this, they are very stable in body fluids, which means that such RNAs can be isolated and analyzed. Since their numbers are small, researchers will have to use very sensitive methods of analysis. However, nothing is impossible here: the sensitivity of nucleic acid sequencing is constantly increasing 27 . Data have been published confirming the promise of this approach in relation to breast cancer 28 , ovarian cancer 29 and a number of other oncological diseases. Analysis of circulating small RNAs in lung cancer patients showed that these RNAs help distinguish between patients with a solitary lung nodule (who do not require therapy) and patients who develop malignant tumor nodules (requiring treatment) 30 .

Article for the competition "bio/mol/text": In recent years, RNA - and especially its "non-classical" varieties - has attracted the attention of biologists around the world. It turned out that regulation with the help of non-coding RNAs is widespread - from viruses and bacteria to humans. The study of the diversity of small bacterial RNA regulators clearly showed their important role in both intermediate metabolism and adaptive responses. This article describes the varieties of small RNAs of bacteria and the mechanisms of regulation carried out with their help. Particular emphasis is placed on the role of these molecules in the vital activity of bacterial agents that cause especially dangerous infections.

RNA: more than just a copy of DNA

Most of the readers of this site know the basic mechanisms of the living cell from school. From the laws of Mendel to state-of-the-art genome sequencing projects, the idea of ​​a master genetic program for the development of an organism, known to professional biologists as central dogma of molecular biology. It states that the DNA molecule acts as a carrier and custodian of genetic information, which through an intermediary - matrix RNA (mRNA), and with the participation of ribosomal (rRNA) and transfer RNA (tRNA) - is realized in the form of proteins. The latter determine the species and individual phenotype.

This state of affairs and the assignment of RNA to the role of a secondary participant in the molecular spectacle persisted in the scientific community until the 1980s. A closer look at RNA was forced by the work of T. Check, who showed that RNA can act as a catalyst for chemical reactions. Previously, it was believed that the acceleration of chemical processes in the cell is the prerogative of enzymes, which are exclusively of a protein nature. The discovery of catalytic activity in RNA had far-reaching consequences - coupled with the earlier theoretical work of K. Woese and , it made it possible to draw a possible picture of prebiotic evolution on our planet. The fact is that since the discovery of the function of a carrier of genetic information in DNA, the dilemma about what appeared earlier in the course of evolution - DNA or the protein necessary for the reproduction of DNA - seemed almost as philosophical (that is, pointless) as the question about the primacy of the appearance of the chicken or the egg. After the discovery of T. Czek, the solution took on quite real outlines - a molecule was found that has the properties of both an information carrier and a biocatalyst (albeit in its infancy). Over time, these studies have grown into a whole direction in biology, studying the emergence of life through the prism of the so-called "RNA world" .

So it became obvious that the ancient world of RNA could be related to the origin and flowering of primary life. Nevertheless, it does not automatically follow from this that RNA in modern organisms is not an archaism adapted to the needs of intracellular molecular systems, but a really important member of the molecular ensemble of the cell. Only the development of molecular methods - in particular, nucleic acid sequencing - showed that RNAs are truly indispensable in the cell, and not only in the form of the canonical trinity "mRNA, rRNA, tRNA". Already the first extensive data on DNA sequencing pointed to a fact that seemed difficult to explain at first - most of it turned out to be non-coding- that is, not carrying information about protein molecules or "standard" RNA. Of course, this can be partially attributed to "genetic garbage" - "turned off" or fragments of the genome that have lost their function. But it seems illogical to keep such a quantity of "dowry" for biological systems trying to use energy economically.

Indeed, more detailed and subtle research methods have made it possible to discover a whole class of RNA regulators of gene expression that partially fill the intergenic space. Even before reading the complete eukaryotic genome sequences in the roundworm C. elegans miRNAs have been isolated - molecules of small length (about 20 nucleotides) that can specifically bind to mRNA regions according to the principle of complementarity. It is easy to guess that in such cases with mRNA it is no longer possible to read information about the encoded proteins: the ribosome simply cannot “run through” such a site that has suddenly become double-stranded. This mechanism for suppressing gene expression, called RNA interference, has already been analyzed on the "biomolecule" in sufficient detail. To date, thousands of microRNA and other non-coding RNA molecules (piRNA , snoRNA , nanoRNA, etc.) have been discovered. In eukaryotes (including humans), they are located in intergenic regions. Their important role in cell differentiation, carcinogenesis, immune response and other processes and pathologies has been established.

Small RNAs are a "Trojan horse" for bacterial proteins

Despite the fact that protein-coding RNAs in bacteria were discovered much earlier than the first similar regulators in eukaryotes, their role in bacterial cell metabolism was veiled to the scientific community for a long time. This is understandable - traditionally, the bacterial cell was considered a more primitive and less mysterious structure for the researcher, the complexity of which cannot be compared with the heap of structures in a eukaryotic cell. Moreover, in bacterial genomes, the content of non-coding information is only a few percent of the total DNA length, reaching a maximum of 40% in some mycobacteria. But, given that microRNAs are found even in viruses, they should play an important regulatory role in bacteria, and even more so.

It turned out that there are quite a lot of small RNA regulators in prokaryotes. Conventionally, all of them can be divided into two groups:

1. RNA molecules that must bind to proteins in order to carry out their function.
2. RNAs that bind complementarily to other RNAs (the majority of known regulatory RNA molecules).

In the first group, small RNAs are isolated, for which protein binding is possible, but not necessary. A well-known example is RNase P (RNAse P), which acts as a ribozyme on a "maturing" tRNA. However, if RNase P can function without a protein component, then for other small RNAs in this group, protein binding is mandatory (and they themselves are, in fact, cofactors). For example, tmRNA activates a complex protein complex, acting as a "master key" for a "stuck" ribosome - if the messenger RNA from which the reading is being performed has come to an end, but the stop codon has not been encountered.

An even more intriguing mechanism for the direct interaction of small RNAs with proteins is also known. Proteins that bind to "traditional" nucleic acids are widespread in any cell. The prokaryotic cell is no exception. For example, its histone-like proteins help to correctly package the DNA strand, and specific repressor proteins have an affinity for the operator region of bacterial genes. It has been shown that these repressors can be inhibited by small RNAs that mimic DNA binding sites native to these proteins. Thus, on the small CsrB RNA (Fig. 1) there are 18 decoy sites that prevent the CsrA repressor protein from reaching its true target, the glycogen operon. By the way, among the repressor proteins “lost” due to such small RNAs, there are regulators of global metabolic pathways, which makes it possible to multiply the inhibitory signal of small RNAs. For example, this is what small RNA 6S does, which “mimics” the protein factor σ 70 . By configurational "deception" by occupying the binding centers of RNA polymerase with the sigma factor, it prohibits the expression of "housekeeping" genes.

Figure 1. Bioinformatically predicted secondary structure of CsrB small RNA from Vibrio cholerae M66-2. Small RNAs are single-stranded molecules, but, as with other RNAs, folding into a stable spatial structure is accompanied by the formation of sites where the molecule hybridizes onto itself. Numerous bends on the structure in the form of open rings are called hairpins. In some cases, the combination of hairpins allows the RNA to act as a "sponge" by non-covalently binding certain proteins. But more often, molecules of this type interfere with DNA or RNA; in this case, the spatial structure of small RNA is disturbed, and new sites of hybridization are formed already with the target molecule. The heat map reflects the probability that the corresponding nucleotide pair will indeed be connected by an intramolecular hydrogen bond; for unpaired sites - the probability of forming hydrogen bonds with any sites within the molecule. Image obtained using the program RNAfold.

Small RNAs of bacteria interfere... and very successfully!

The mechanism by which the regulators of the second group act is, in general, similar to that of eukaryotic regulatory RNAs - this is the same RNA interference by hybridization with mRNA, only the chains of small RNAs themselves are often more authentic - up to several hundred nucleotides ( cm. rice. one). As a result, due to small RNA, ribosomes cannot read information from mRNA. Although often, it seems, it does not even come to this: the resulting “small RNA-mRNA” complexes become the target of RNases (RNase P type).

The compactness and packing density of the prokaryotic genome makes itself felt: if in eukaryotes most of the regulatory RNAs are recorded in separate (most often non-coding protein) loci, then many small bacterial RNAs can be encoded in the same DNA region as the repressed gene, but on the opposite side. chains! Such RNAs are called cis-coded(antisense), and small RNAs lying at some distance from the repressed DNA region - trans-coded. Apparently, the arrangement of cis-RNAs can be considered a triumph of ergonomics: they can be read from the opposite DNA strand at the moment of its unwinding simultaneously with the target transcript, which makes it possible to finely control the amount of synthesized protein.

Small trans RNAs evolve independently of the target mRNA, and the sequence of the regulator changes more strongly as a result of mutations. It is possible that such an arrangement is only “on hand” for the bacterial cell, since small RNA acquires activity against previously uncharacteristic targets, which reduces the time and energy costs for the creation of other regulators. On the other hand, selection pressure prevents the trans-small RNA from mutating too much, as it will lose activity. However, most trans-small RNAs require a helper, the Hfq protein, to hybridize with messenger RNA. Apparently, otherwise incomplete complementarity of small RNA can create problems for binding to the target.

Apparently, the potential mechanism of regulation based on the principle of "one small RNA - many targets" helps to integrate the bacterial metabolic networks, which is extremely necessary under conditions of a short unicellular life. One can continue to speculate on the topic and assume that trans-encoded small RNAs are used to send expression "instructions" from functionally related but physically distant loci. The need for this kind of genetic “roll call” logically explains the large number of small RNAs found in pathogenic bacteria. For example, several hundred small RNAs were found in the record holder for this indicator - cholera vibrio ( Vibrio cholerae). This is a microorganism that can survive in the surrounding aquatic environment (both fresh and salty), and on aquatic mollusks, and in fish, and in the human intestine - here you can’t do without complex adaptation with the help of regulatory molecules!

CRISPR for bacterial health

Small RNAs have also found application in solving another vital problem for bacteria. Even the most malicious pathogenic cocci and bacilli can be powerless in the face of the danger posed by special viruses - bacteriophages that can exterminate the bacterial population with lightning speed. Multicellular organisms have a specialized system for protection against viruses - immune, by means of cells and the substances they secrete, protecting the body from intruders (including those of a viral nature). The bacterial cell is a loner, but it is not as vulnerable as it might seem at first glance. The custodians of the recipes for maintaining the antiviral immunity of bacteria are the loci CRISPR- cluster regular-discontinuous short palindromic repeats ( clustered regularly interspaced short palindromic repeats) (Fig. 2; ). In prokaryotic genomes, each CRISPR cassette is represented by a leader sequence several hundred nucleotides long, followed by a series of 2–24 (sometimes up to 400) repeats separated by spacer regions similar in length but unique in nucleotide sequence. The length of each spacer and repeat does not exceed one hundred base pairs.

Figure 2. CRISPR locus and processing of its corresponding small RNA to a functional transcript. In the genome CRISPR- the cassette is represented by spacers interleaved (in the figure they are indicated as sp), partially homologous regions of phage DNA, and repeats ( By) 24–48 bp long, showing dyadic symmetry. In contrast to repeats, spacers within the same locus are the same in length (in different bacteria this can be 20–70 nucleotides), but differ in nucleotide sequence. The “-spacer-repeat-” sections can be quite long and consist of several hundred links. The entire structure is flanked on one side by the leader sequence ( LP, several hundred base pairs). Nearby are Cas-genes ( C RISPR-as sociated) organized into an operon. Proteins read from them perform a number of auxiliary functions, providing processing of the transcript read from CRISPR-locus, its successful hybridization with the phage DNA target, incorporation of new elements into the locus, etc. The CrRNA formed as a result of multi-stage processing hybridizes with a DNA region (lower part of the figure) injected by the phage into the bacterium. This silences the transcription machine of the virus and stops it from multiplying in the prokaryotic cell.

Detailed mechanism of the origin of everything CRISPR-locus is yet to be studied. But to date, a schematic diagram of the emergence of spacers, the most important structures in its composition, has been proposed. It turns out that "bacteria hunters" are beaten by their own weapons - nucleic acids, or rather, "trophy" genetic information received by bacteria from phages in previous battles! The fact is that not all phages that enter a bacterial cell turn out to be fatal. The DNA of such phages (possibly related to temperate ones) is cut by special Cas proteins (their genes flank CRISPR) into small fragments. Some of these fragments will be embedded in CRISPR loci of the "host" genome. And when the phage DNA re-enters the bacterial cell, it meets small RNA from CRISPR-locus, at that moment expressed and processed by Cas proteins. This is followed by the inactivation of viral genetic information according to the mechanism of RNA interference already described above.

From the hypothesis of the formation of spacers, it is not clear why repeats are needed between them, within the same locus slightly different in length, but almost identical in sequence? There is a wide scope for imagination here. Perhaps, without repetitions, it would be problematic to split genetic data into semantic fragments, similar to sectors on a computer hard drive, and then the transcription machine would have access to strictly defined areas CRISPR-locus would become difficult? Or maybe repeats simplify recombination processes when new elements of phage DNA are inserted? Or are they "punctuation marks" that are indispensable for CRISPR processing? Be that as it may, the biological reason explaining the behavior of the bacterial cell in the manner of Gogol's Plyushkin will be found in due time.

CRISPR, being a "chronicle" of the relationship between a bacterium and a phage, can be used in phylogenetic studies. Thus, the recently carried out typing according to CRISPR made it possible to look at the evolution of individual strains of the plague microbe ( Yersinia pestis). Exploring them CRISPR- “pedigrees” shed light on the events of half a thousand years ago, when strains entered Mongolia from the territory now belonging to China. But not for all bacteria, and, in particular, pathogens, this method is applicable. Despite recent evidence of predicted CRISPR-processing proteins in tularemia ( Francisella tularensis) and cholera, CRISPR itself, if present in their genome, is not numerous. Perhaps phages, given their positive contribution to the acquisition of virulence by pathogenic representatives of the bacterial kingdom, are not so harmful and dangerous as to be protected from them using CRISPR? Or are the viruses that attack these bacteria too diverse, and the strategy of “interfering” RNA immunity against them is fruitless?

Figure 3. Some mechanisms of operation of riboswitches. Riboswitches (riboswitches) are built into messenger RNA, but they are distinguished by a large freedom of conformational behavior depending on specific ligands, which gives reason to consider riboswitches as independent units of small RNAs. A change in the conformation of the expression platform affects the ribosome entry site on mRNA ( RBS), and, as a result, determines the availability of all mRNA for reading. Riboswitches are to a certain extent similar to the operator domain in the classical model lac-operon - but only aptameric regions are usually regulated by low molecular weight substances and switch the work of the gene at the level of mRNA, not DNA. a - In the absence of ligands, riboswitches btuB (cobalamin transporter) and thiM (thiamine pyrophosphate-dependent), which carry out non-nucleolytic repression of mRNA, are “switched on” ( ON) and let the ribosome do its thing. Binding of a ligand to a riboswitch ( OFF-position) leads to the formation of a hairpin, which makes this site inaccessible to the ribosome. b - Lysine Riboswitch lysC in the absence of a ligand is also included ( ON). Turning off the riboswitch blocks the ribosome from accessing the mRNA. But unlike the riboswitches described above, in the lysine switch, when the switch is turned off, the area cut by a special RNase complex ( degradesome), and all mRNA is utilized, breaking up into small fragments. Riboswitch repression in this case is called nucleolytic ( nucleolytic) and is irreversible because, unlike the example ( a ), reverse switching (back to ON) is no longer possible. It is important to note that the utilization of a group of “unnecessary” mRNAs can be achieved in this way: a riboswitch is similar to a part of a children's construction kit, and a whole group of functionally related template molecules can have similar switches in structure.

Riboswitch - sensor for bacteria

So, there are protein-associating small RNAs, there are small RNAs that interfere with the bacteria's own mRNA, as well as RNA captured by bacteria from viruses and suppress phage DNA. Is it possible to imagine any other mechanism of regulation with the help of small RNAs? It turns out yes. If we analyze the above, it will be found that in all cases of antisense regulation, interference of small RNA and the target is observed as a result of hybridization of two individual molecules. Why not arrange small RNA within the transcript itself? Then it is possible, by changing the conformation of such a "mishandled cossack" inside mRNA, to change the availability of the entire template for reading during translation or, which is even more expedient energetically, to regulate mRNA biosynthesis, i.e. transcription!

Such structures are widely present in bacterial cells and are known as riboswitches ( riboswitch). They are located before the start of the coding part of the gene, at the 5' end of the mRNA. Conditionally, two structural motifs can be distinguished in the composition of riboswitches: aptamer region, responsible for binding to the ligand (effector), and expression platform, providing regulation of gene expression through the transition of mRNA to alternative spatial structures. For example, such a switch ("off" type) is used to operate lysine operon: with an excess of lysine, it exists in the form of a “tangled” spatial structure that blocks reading from the operon, and with a shortage of lysine, the riboswitch “unwinds” and proteins necessary for lysine biosynthesis are synthesized (Fig. 3).

The described schematic diagram of the riboswitch device is not a canon, there are options. A curious "switching" tandem riboswitch was found in Vibrio cholerae: the expression platform is preceded by two at once aptamer sites. Obviously, this provides greater sensitivity and a smoother response to the appearance of another amino acid in the cell - glycine. Possibly, the “double” riboswitch in the anthrax genome is indirectly involved in the high survival rate of the bacterium ( Bacillus anthracis). It reacts to the compound that is vital for this microbe - thiamine pyrophosphate, which is part of the minimal environment.

In addition to switching metabolic pathways depending on the “menu” available to the bacterial cell, riboswitches can be sensors of bacterial homeostasis. Thus, they were seen in the regulation of gene availability for reading when the functioning of the translational system inside the cell is disrupted (for example, such signals as the appearance of “uncharged” tRNAs and “faulty” (stalled) ribosomes), or when environmental factors change (for example, temperature rise). ) .

Don't need proteins, give us RNA!

So what does the presence of such a variety of small RNA regulators within a bacterium mean? Does this indicate a rejection of the concept, when the main "managers" are proteins, or are we seeing another fashion trend? Apparently, neither one nor the other. Of course, some small RNAs are global regulators of metabolic pathways, such as the mentioned CsrB, which, together with CsrC, is involved in the regulation of organic carbon storage. But, taking into account the principle of duplication of functions in biological systems, small RNAs of bacteria can be compared more with an “anti-crisis manager” than with a general director. So, in conditions when for the survival of a microorganism it is necessary fast reconfigure intracellular metabolism, their regulatory role may turn out to be decisive and more effective than that of proteins with similar functions. Thus, RNA regulators are more likely responsible for rapid response, which is less stable and reliable than in the case of proteins: we should not forget that small RNA maintains its 3D structure and is retained on the inhibited matrix by weak hydrogen bonds.

Indirect confirmation of these theses can be the already mentioned small RNAs of cholera vibrio. For this bacterium, entering the human body is not a desirable goal, but, apparently, an emergency. The production of toxins and the activation of other pathways associated with virulence in this case is just a defensive reaction to the aggressive opposition of the environment and cells of the body to “outsiders”. The "rescuers" here are small RNAs - for example, Qrr, which help the vibrio to modify the survival strategy under stressful conditions, changing the collective behavior. This hypothesis can also be indirectly confirmed by the discovery of small RNA VrrA, which is actively synthesized when vibrios are present in the body and suppresses the production of Omp membrane proteins. “Hidden” membrane proteins in the initial phase of infection may help to avoid a powerful immune response from the human body (Fig. 4).

Figure 4. Small RNAs in the implementation of the pathogenic properties of Vibrio cholerae. a - Vibrio cholerae feels good and reproduces well in the aquatic environment. The human body is probably not the main ecological niche for this microbe. b - Getting through the water or food route of transmission of infection into an aggressive environment - the human small intestine - vibrios in terms of organization of behavior begin to resemble a pseudo-organism, the main task of which is to restrain the immune response and create a favorable environment for colonization. Membrane vesicles are of great importance in coordinating actions within the bacterial population and their interaction with the body. Until the end, unexplored environmental factors in the intestine are signals for the expression of small RNAs in vibrios (for example, VrrA). As a result, the mechanism of formation of vesicles is triggered, which are non-immunogenic with a low number of vibrio cells in the intestine. In addition to the described effect, small RNAs help to “hide” Omp membrane proteins that are potentially provocative for the human immune system. With the indirect participation of small RNAs Qrr1-4, intensive production of cholera toxin is triggered (not shown in the figure), which complements the spectrum of adaptive reactions of cholera vibrio. in - Already after a few hours, the number of bacterial cells increases, and the pool of small VrrA RNAs decreases, which probably leads to the exposure of membrane proteins. The number of "empty" vesicles also gradually decreases, and at this stage they are replaced by immunogenic ones delivered to enterocytes. Apparently, this is part of the "plan" for the implementation of a complex signal, the meaning of which is to provoke the evacuation of vibrios from the human body. NB: the ratio of the sizes of bacterial cells and enterocytes is not observed.

It is interesting to see how our understanding of small RNA regulators will change when new data are obtained on the RNAseq platforms, including on free-living and non-culturable forms. Recent work using "deep sequencing" has already yielded unexpected results, indicating the presence of miRNA-like molecules in mutant streptococci. Of course, such data need to be carefully rechecked, but, be that as it may, it can be said with confidence that the study of small RNAs in bacteria will bring many surprises.

Thanks

The original ideas and compositional design when creating the title figure, as well as figure 4, belong to E.A. The presence of figure 2 in the article is the merit of the associate professor of the department. zoology SFedU G.B. Bakhtadze. He also carried out scientific proofreading and revision of the title figure and figure 4. The author expresses his deep gratitude to them for their patience and creative approach to the matter. Special thanks to my colleague, Senior Researcher lab. Biochemistry of Microbes of the Rostov Anti-Plague Institute Sorokin V.M. for discussion of the text of the article and for valuable comments.

Literature

1. Carl Woese (1928–2012) ;;. 80 , 1148-1154;
2. R. R. Breaker. (2012). Riboswitches and the RNA World. Cold Spring Harbor Perspectives in Biology. 4 , a003566-a003566;
3. J. Patrick Bardill, Brian K. Hammer. (2012). Non-coding sRNAs regulate virulence in the bacterial pathogen Vibrio cholerae . RNA Biology. 9 , 392-401;
4. Heon-Jin Lee, Su-Hyung Hong. (2012). Analysis of microRNA-size, small RNAs in Streptococcus mutans by deep sequencing . FEMS Microbiol Lett. 326 , 131-136;
5. M.-P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, D. A. Lafontaine. (2012). Dual-acting riboswitch control of translation initiation and mRNA decay . Proceedings of the National Academy of Sciences. 109 , E3444-E3453.

The metaphor underlying the name of the phenomenon of RNA interference refers to the experiment with petunia, when the genes for the synthetase of pink and purple pigments artificially introduced into the plant did not increase the color intensity, but, on the contrary, reduced it. Similarly, in "ordinary" interference, the superposition of two waves can lead to mutual "quenching".

In a living cell, the flow of information between the nucleus and the cytoplasm never dries up, but understanding all its "twists" and deciphering the information encoded in it is a truly titanic task. One of the most important breakthroughs in biology of the last century can be considered the discovery of information (or template) RNA molecules (mRNA or mRNA), which serve as intermediaries that carry information "messages" from the nucleus (from chromosomes) to the cytoplasm. The decisive role of RNA in protein synthesis was predicted as early as 1939 in the work of Torbjörn Caspersson, Jean Brachet and Jack Schultz, and in 1971 George Marbaix launched the synthesis of hemoglobin in oocytes frogs by injecting the first isolated rabbit messenger RNA encoding this protein.

In 1956-57 in the Soviet Union, A. N. Belozersky and A. S. Spirin independently proved the existence of mRNA, and also found out that the bulk of RNA in a cell is by no means matrix, but ribosomal RNA (rRNA). Ribosomal RNA - the second "main" type of cellular RNA - forms the "skeleton" and functional center of ribosomes in all organisms; it is rRNA (and not proteins) that regulates the main stages of protein synthesis. At the same time, the third “main” type of RNA, transfer RNA (tRNA), was described and studied, which, in combination with the other two, mRNA and rRNA, form a single protein-synthesizing complex. According to the rather popular hypothesis of the "RNA world", it was this nucleic acid that lay at the very origins of life on Earth.

Due to the fact that RNA is much more hydrophilic compared to DNA (due to the replacement of deoxyribose by ribose), it is more labile and can move relatively freely in the cell, and hence deliver short-lived replicas of genetic information (mRNA) to the place where protein synthesis. However, it is worth noting the “inconvenience” associated with this - RNA is very unstable. It is stored much worse than DNA (even inside the cell) and degrades at the slightest change in conditions (temperature, pH). In addition to their "own" instability, a large contribution belongs to ribonucleases (or RNases) - a class of RNA-cleaving enzymes, very stable and "ubiquitous" - even the skin of the experimenter's hands contains enough of these enzymes to cross out the entire experiment. Because of this, working with RNA is much more difficult than with proteins or DNA - the latter can generally be stored for hundreds of thousands of years with little or no damage.

Fantastic accuracy during work, tridistillate, sterile gloves, disposable laboratory glassware - all this is necessary to prevent RNA degradation, but compliance with such standards was not always possible. Therefore, for a long time, short “fragments” of RNA, which inevitably polluted solutions, were simply ignored. However, over time, it became clear that, despite all efforts to maintain the sterility of the working area, “debris” naturally continued to be detected, and then it turned out that thousands of short double-stranded RNAs are always present in the cytoplasm, performing quite specific functions, and absolutely necessary for normal development cells and organisms.

Principle of RNA interference

Today, the study of small regulatory RNAs is one of the most rapidly developing areas of molecular biology. It was found that all short RNAs perform their functions on the basis of a phenomenon called RNA interference (the essence of this phenomenon is the suppression of gene expression at the stage of transcription or translation with the active participation of small RNA molecules). Very schematically, the mechanism of RNA interference is shown in Fig. 1:

Rice. 1. Fundamentals of RNAi
Double-stranded RNA molecules (dsRNA) are not characteristic of normal cells, but they are an essential step in the life cycle of many viruses. A special Dicer protein, having found dsRNA in a cell, “cuts” it into small fragments. The antisense chain of such a fragment, which can already be called short interfering RNA (siRNA, from siRNA - small interference RNA), is bound by a protein complex called RISC (RNA-induced silencing complex), the central element of which is an endonuclease of the Argonaute family. Binding to siRNA activates RISC and triggers a search for DNA and RNA molecules in the cell that are complementary to the “template” siRNA. The fate of such molecules is to be destroyed or inactivated by the RISC complex.

Summing up, short “trimmings” of foreign (including intentionally introduced) double-stranded RNA serve as a “template” for a large-scale search and destruction of complementary mRNAs (and this is equivalent to suppression of the expression of the corresponding gene), not only in one cell, but also in neighboring. For many organisms - protozoa, molluscs, worms, insects, plants - this phenomenon is one of the main ways of immune defense against infections.

In 2006, Andrew Fire and Craig Mello received the Nobel Prize in Physiology or Medicine "for their discovery of the phenomenon of RNA interference - the mechanism of gene silencing with the participation of dsRNA". Although the phenomenon of RNA interference itself was described long before (back in the early 1980s), it was the work of Fire and Mello that outlined the regulatory mechanism of small RNAs and outlined the hitherto unknown area of ​​molecular research. Here are the main results of their work:

• In RNA interference, it is the mRNA (and no other) that is cleaved;
• Double-stranded RNA acts (causes cleavage) much more efficiently than single-stranded. These two observations predicted the existence of a specialized system mediating the action of dsRNA;
• dsRNA, complementary to a section of mature mRNA, causes cleavage of the latter. This indicated the cytoplasmic localization of the process and the presence of a specific endonuclease;
• A small amount of dsRNA (several molecules per cell) is sufficient to completely "turn off" the target gene, which indicates the existence of a cascade mechanism of catalysis and/or amplification.

These results laid the foundation for a whole area of ​​modern molecular biology - RNA interference - and determined the vector of work of many research groups around the world for more than a dozen years. To date, three large groups of small RNAs have been discovered that play in the molecular field for the “RNA interference team”. Let's get to know them in more detail.

Player #1 - Short Interfering RNAs

The specificity of RNA interference is determined by short interfering RNAs (siRNAs) - small double-stranded RNA molecules with a well-defined structure (see Fig. 2).

siRNAs are evolutionarily the earliest, and are most widely distributed in plants, unicellular organisms, and invertebrates. In normal vertebrates, siRNAs are practically not found, because they were supplanted by later “models” of short RNAs (see below).

siRNAs - "templates" for searching in the cytoplasm and destroying mRNA molecules - have a length of 20–25 nucleotides and a "special sign": 2 unpaired nucleotides at the 3'-ends and phosphorylated 5'-ends. Anti-sense siRNA is capable (not by itself, of course, but with the help of a RISC complex) to recognize mRNA and specifically cause its degradation: the cut of the target mRNA always occurs exactly in the place complementary to 10 and 11 nucleotides of the anti-sense siRNA strand.

Rice. 2. Mechanism of “interference” between mRNA and siRNA
"Interfering" short RNA molecules can both enter the cell from the outside, and "cut" already in place from longer double-stranded RNA. The main protein required for "cutting" dsRNA is Dicer endonuclease. “Switching off” the gene by the interference mechanism is carried out by siRNA together with the RISC protein complex, which consists of three proteins - Ago2 endonuclease and two auxiliary proteins PACT and TRBP. Later, it was found that Dicer and RISC complexes can use not only dsRNA but also single-stranded RNA, which forms a double-stranded hairpin, as well as ready-made siRNA (the latter bypasses the “cutting” stage and immediately binds to RISC) as a “seed”.

The functions of siRNAs in invertebrate cells are quite diverse. The first and main one is immune protection. The "traditional" immune system (lymphocytes + leukocytes + macrophages) is present only in complex multicellular organisms. In unicellular organisms, invertebrates and plants (which either do not have such a system, or it is in its infancy), immune defense is built on the basis of RNA interference. Immunity based on RNA interference does not need complex organs of "training" of the precursors of immune cells (spleen, thymus); at the same time, the variety of theoretically possible short RNA sequences (421 variants) is correlated with the number of possible protein antibodies in higher animals. In addition, siRNAs are synthesized on the basis of the “hostile” RNA that infected the cell, which means, unlike antibodies, they are immediately “sharpened” for a specific type of infection. And although protection based on RNA interference does not work outside the cell (at least, there are no such data yet), it provides intracellular immunity more than satisfactorily.

First of all, siRNA creates antiviral immunity by destroying the mRNA or genomic RNA of infectious organisms (for example, this is how siRNA was discovered in plants). The introduction of viral RNA causes a powerful amplification of specific siRNAs based on a seed molecule - the viral RNA itself. In addition, siRNAs suppress the expression of various mobile genetic elements (MGEs), which means that they also provide protection against endogenous “infections”. Mutations in the genes of the RISC complex often lead to increased genome instability due to high MGE activity; siRNA can be a limiter of the expression of its own genes, triggering in response to their overexpression. Regulation of the work of genes can occur not only at the level of translation, but also during transcription - through methylation of genes at the H3 histone.

In modern experimental biology, the importance of RNA interference and short RNAs cannot be overestimated. The technology of "turning off" (or knocking down) individual genes in vitro (on cell cultures) and in vivo (on embryos) has been developed, which has already become the de facto standard in the study of any gene. Sometimes, even in order to establish the role of individual genes in some process, they systematically “turn off” all genes in turn.

Pharmacists have also become interested in the possibility of using siRNA, since the ability to regulate the work of individual genes promises unheard-of prospects in the treatment of a host of diseases. The small size and high specificity of action promise high efficacy and low toxicity of siRNA-based drugs; however, it has not yet been possible to solve the problem of siRNA delivery to diseased cells in the body due to the fragility and fragility of these molecules. And although now dozens of teams are trying to find a way to direct these “magic bullets” exactly at the target (inside diseased organs), they have not yet achieved visible success. In addition, there are other difficulties. For example, in the case of antiviral therapy, the high selectivity of the action of siRNA can be a disservice - since viruses quickly mutate, the modified strain will very quickly lose sensitivity to the siRNA selected at the beginning of therapy: it is known that the replacement of just one nucleotide in siRNA leads to a significant decrease in interference effect.

At this point, it is worth recalling once again that siRNAs have only been found in plants, invertebrates, and unicellular organisms; Although homologues of proteins for RNA interference (Dicer, RISC complex) are also present in higher animals, siRNAs have not been detected by conventional methods. What a surprise it was when artificially introduced synthetic siRNA analogues caused a strong specific dose-dependent effect in mammalian cell cultures! This meant that in vertebrate cells, RNA interference was not replaced by more complex immune systems, but evolved along with organisms, turning into something more “advanced”. Consequently, in mammals it was necessary to look not for exact analogs of siRNAs, but for their evolutionary successors.

Player #2 - miRNA

Indeed, on the basis of the evolutionarily rather ancient mechanism of RNA interference, more developed organisms have developed two specialized systems for controlling the work of genes, each using its own group of small RNAs - microRNA (microRNA) and piRNA (piRNA, Piwi-interacting RNA). Both systems appeared in sponges and coelenterates and evolved together with them, displacing siRNA and the mechanism of "naked" RNA interference. Their role in providing immunity is declining, since this function has been taken over by more advanced mechanisms of cellular immunity, in particular, the interferon system. However, this system is so sensitive that it also works on siRNA itself: the appearance of small double-stranded RNAs in a mammalian cell triggers an “alarm signal” (activates the secretion of interferon and causes the expression of interferon-dependent genes, which blocks all translation processes entirely). In this regard, the mechanism of RNA interference in higher animals is mediated mainly by microRNA and piRNA, single-stranded molecules with a specific structure that are not detected by the interferon system.

As the genome became more complex, miRNAs and piRNAs became increasingly involved in the regulation of transcription and translation. Over time, they evolved into an additional, precise and subtle system of genome regulation. Unlike siRNAs, microRNAs and piRNAs (discovered in 2001, see Fig. 3, A-B) are not produced from foreign double-stranded RNA molecules, but are initially encoded in the genome of the host organism.

The microRNA precursor is transcribed from both strands of genomic DNA by RNA polymerase II, resulting in an intermediate form, pri-miRNA, that carries the features of conventional mRNA, the m7G cap and polyA tail. This precursor forms a loop with two single-stranded “tails” and several unpaired nucleotides in the center (Fig. 3A). Such a loop undergoes two-stage processing (Fig. B): first, Drosha endonuclease cuts off single-stranded RNA “tails” from the hairpin, after which the cut hairpin (pre-microRNA) is exported to the cytoplasm, where it is recognized by Dicer, which makes two more cuts (a double-stranded region is cut out). , indicated by color in Fig. 3A). In this form, mature miRNA, similarly to siRNA, is included in the RISC complex.

The mechanism of action of many miRNAs is similar to that of siRNAs: a short (21–25 nucleotides) single-stranded RNA in the RISC protein complex binds with high specificity to a complementary site in the 3'-untranslated region of the target mRNA. Binding results in cleavage of the mRNA by the Ago protein. However, the activity of microRNAs (compared to siRNAs) is already more differentiated: if the complementarity is not absolute, the target mRNA may not be degraded, but only reversibly blocked (there will be no translation). The same RISC complex can also use artificially introduced siRNAs. This explains why siRNAs, made by analogy with protozoa, are also active in mammals.

Thus, we can supplement the illustration of the mechanism of action of RNA interference in higher (bilaterally symmetrical) organisms by combining in one figure the scheme of action of miRNAs and biotechnologically introduced siRNAs (Fig. 3C).

Rice. 3A: Structure of a double-stranded miRNA precursor molecule
Main features: the presence of conserved sequences that form a hairpin; the presence of a complementary copy (microRNA*) with two “extra” nucleotides at the 3’-end; specific sequence (2–8 bp) that forms the recognition site for endonucleases. The miRNA itself is highlighted in red - that is what Dicer cuts out.

Rice. 3B: General mechanism of miRNA processing and realization of its activity

Rice. 3B: Generalized scheme of the action of artificial miRNAs and siRNAs
Artificial siRNAs are introduced into the cell using specialized plasmids (targeting siRNA vector).

Functions of miRNA

Physiological functions of miRNAs are extremely diverse; in fact, they act as the main non-protein regulators of ontogeny. miRNAs do not cancel, but complement the "classical" scheme of gene regulation (inductors, suppressors, chromatin compaction, etc.). In addition, the synthesis of microRNAs themselves is regulated in a complex way (certain pools of microRNAs can be turned on by interferons, interleukins, tumor necrosis factor α (TNF-α), and many other cytokines). As a result, a multi-level network of setting up an "orchestra" of thousands of genes, amazing in its complexity and flexibility, emerges, but this is not the end of the matter.

miRNAs are more “universal” than siRNAs: “ward” genes do not have to be 100% complementary – regulation is also carried out with partial interaction. Today, one of the hottest topics in molecular biology is the search for microRNAs, which act as alternative regulators of known physiological processes. For example, miRNAs involved in the regulation of the cell cycle and apoptosis in plants, Drosophila and nematodes have already been described; in humans, miRNAs regulate the immune system and hematopoietic stem cell development. The use of technologies based on biochips (micro-array screening) has shown that entire pools of small RNAs are turned on and off at different stages of cell life. For biological processes, dozens of specific microRNAs have been identified, the expression level of which under certain conditions changes thousands of times, emphasizing the exceptional controllability of these processes.

Until recently, it was believed that miRNAs only suppress - in whole or in part - the work of genes. However, recently it turned out that the action of miRNAs can radically differ depending on the state of the cell! In an actively dividing cell, microRNA binds to a complementary sequence in the 3'-site of mRNA and inhibits protein synthesis (translation). However, in a state of rest or stress (for example, when growing on a poor medium), the same event leads to the opposite effect - an increase in the synthesis of the target protein!

Evolution of miRNA

The number of microRNA varieties in higher organisms has not yet been fully established - according to some data, it exceeds 1% of the number of protein-coding genes (in humans, for example, they talk about 700 microRNAs, and this number is constantly growing). microRNAs regulate the activity of about 30% of all genes (targets for many of them are not yet known), and there are both ubiquitous and tissue-specific molecules - for example, one such important pool of microRNAs regulates the maturation of blood stem cells.

The wide expression profile in different tissues of different organisms and the biological abundance of miRNAs indicate an evolutionarily ancient origin. For the first time, miRNAs were found in nematodes, and for a long time it was believed that these molecules appear only in sponges and coelenterates; however, later they were also discovered in unicellular algae. Interestingly, as organisms become more complex, the number and heterogeneity of the microRNA pool also increase. This indirectly indicates that the complexity of these organisms is provided, in particular, by the functioning of miRNAs. Possible evolution of miRNA is shown in Fig.4.

Rice. 4. Diversity of miRNAs in different organisms
The higher the organization of the organism, the more miRNAs are found in it (the number in brackets). Species with single miRNAs are highlighted in red. According to .

A clear evolutionary relationship can be drawn between siRNA and microRNA based on the following facts:

• the action of both species is interchangeable and mediated by homologous proteins;
• siRNAs introduced into mammalian cells specifically “turn off” the necessary genes (despite some activation of interferon protection);
• miRNAs are found in more and more ancient organisms.

These and other data suggest the origin of both systems from a common "ancestor". It is also interesting to note that "RNA" immunity as an independent precursor of protein antibodies confirms the theory of the origin of the first life forms based on RNA, not proteins (recall that this is the favorite theory of Academician A. S. Spirin).

While there were only two "players" in the arena of molecular biology - siRNA and microRNA - the main "purpose" of RNA interference seemed completely clear. Indeed: a set of homologous short RNAs and proteins in different organisms performs similar actions; as organisms become more complex, so does their functionality.

However, in the process of evolution, nature created another, evolutionarily latest and highly specialized system based on the same successful principle of RNA interference. We are talking about piRNA (piRNA, from Piwi-interaction RNA).

The more complex the genome is organized, the more developed and adapted the organism (or vice versa? ;-). However, the increase in the complexity of the genome has a downside: the complex genetic system becomes unstable. This leads to the need for mechanisms responsible for maintaining the integrity of the genome - otherwise spontaneous "mixing" of DNA will simply disable it. Mobile genetic elements (MGEs), one of the main factors of genome instability, are short unstable regions that can autonomously transcribe and migrate throughout the genome. Activation of such transposable elements leads to multiple DNA breaks in chromosomes, which are fraught with lethal consequences.

The number of MGEs increases non-linearly with genome size, and their activity must be controlled. To do this, animals, already starting with coelenterates, use the same phenomenon of RNA interference. This function is also performed by short RNAs, however, not those that have already been discussed, but their third type, piRNAs.

"Portrait" of piRNA

piRNAs are short molecules 24-30 nucleotides long, encoded in the centromeric and telomeric regions of the chromosome. The sequences of many of them are complementary to known mobile genetic elements, but there are many other piRNAs that coincide with regions of working genes or with genome fragments whose functions are unknown.

piRNAs (as well as microRNAs) are encoded in both strands of genomic DNA; they are very variable and diverse (up to 500,000 (!) species in one organism). Unlike siRNAs and microRNAs, they are formed by a single strand with a characteristic feature - uracil (U) at the 5' end and a methylated 3' end. There are other differences as well:

• Unlike siRNAs and miRNAs, they do not require Dicer processing;
• piRNA genes are active only in germ cells (during embryogenesis) and surrounding endothelial cells;
• The protein composition of the piRNA system is different - these are Piwi class endonucleases (Piwi and Aub) and a separate variety of Argonaute - Ago3.

The processing and activity of piRNAs are still poorly understood, but it is already clear that the mechanism of action is completely different from other short RNAs - today a ping-pong model of their work has been proposed (Fig. 5 A, B).

Ping-pong mechanism of piRNA biogenesis

Rice. 5A: Cytoplasmic part of piRNA processing
PiRNA biogenesis and activity is mediated by the Piwi endonuclease family (Ago3, Aub, Piwi). piRNA activity is mediated by both single-stranded piRNA molecules, sense and anti-sense, each of which associates with a specific Piwi endonuclease. piRNA recognizes the complementary region of the transposon mRNA (blue strand) and cuts it out. This not only inactivates the transposon, but also creates a new piRNA (linked to Ago3 via Hen1 methylase methylation of the 3' end). Such piRNA, in turn, recognizes mRNA with transcripts of the piRNA precursor cluster (red strand) - in this way the cycle closes and the desired piRNA is produced again.

Rice. 5B: piRNA in the nucleus
In addition to Aub endonuclease, Piwi endonuclease can also bind antisense piRNA. After binding, the complex migrates to the nucleus, where it causes degradation of complementary transcripts and chromatin rearrangement, causing suppression of transposon activity.

piRNA functions

The main function of piRNA is the suppression of MGE activity at the level of transcription and translation. It is believed that piRNAs are active only during embryogenesis, when unpredictable shuffling of the genome is especially dangerous and can lead to the death of the embryo. This is logical - when the immune system has not yet worked, the cells of the embryo need some simple but effective protection. From external pathogens, the embryo is reliably protected by the placenta (or egg shell). But besides this, defense is also needed from endogenous (internal) viruses, primarily MGE.

This role of piRNA has been confirmed by experience – “knockout” or mutations of the Ago3, Piwi, or Aub genes lead to serious developmental disorders (and a sharp increase in the number of mutations in the genome of such an organism), and also cause infertility due to impaired development of germ cells.

Distribution and evolution of piRNA

The first piRNAs are already found in sea anemones and sponges. Plants, apparently, went the other way - Piwi proteins were not found in them, and the role of a "muzzle" for transposons is performed by the Ago4 endonuclease and siRNA.

In higher animals, including humans, the piRNA system is very well developed, but it can only be found in embryonic cells and in the amniotic endothelium. Why the distribution of piRNA in the body is so limited remains to be seen. It can be assumed that, like any powerful weapon, piRNA is useful only in very specific conditions (during fetal development), and in an adult organism, their activity will do more harm than good. Still, the number of piRNAs is an order of magnitude greater than the number of known proteins, and it is difficult to predict the nonspecific effects of piRNAs in mature cells.

Conclusion

In conclusion, I would like to give a table illustrating the evolution of the protein apparatus involved in RNA interference (Fig. 6). It can be seen that protozoa have the most developed siRNA system (protein families Ago, Dicer), and with the complication of organisms, the emphasis shifts to more specialized systems: the number of protein isoforms for microRNA (Drosha, Pasha) and piRNA (Piwi, Hen1) increases. At the same time, the diversity of enzymes mediating the action of siRNA decreases.

Rice. 6. Variety of proteins involved in RNA interference and
The numbers indicate the number of proteins in each group. Elements characteristic of siRNA and microRNA are highlighted in blue, and proteins associated with piRNA are highlighted in red. According to .

The phenomenon of RNA interference began to be used by the simplest organisms. Based on this mechanism, nature created a prototype of the immune system, and as organisms become more complex, RNA interference becomes an indispensable regulator of genome activity. Two different mechanisms plus three types of short RNA (see summary table) - as a result, we see thousands of subtle regulators of various metabolic and genetic pathways. This striking picture illustrates the versatility and evolutionary adaptation of molecular biological systems. Short RNAs again prove that there are no “little things” inside the cell - there are only small molecules, the full significance of whose role we are just beginning to understand.

True, such a fantastic complexity speaks rather about the fact that evolution is “blind” and operates without a pre-approved “master plan”.

Literature

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2. Spirin A. S. (2001). Protein Biosynthesis, the RNA World and the Origin of Life. Bulletin of the Russian Academy of Sciences 71, 320-328;
3. Elements: "Complete mitochondrial genomes of extinct animals can now be extracted from hair";
4. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-311;
5. Biomolecule: "MicroRNA found for the first time in a unicellular organism";
6. Covey S., Al-Kaff N., Lángara A., Turner D. (1997). Plants combat infection by gene silencing. Nature 385, 781-782;
7. Biomolecule: "Molecular Double Dealing: Human Genes Work for the Influenza Virus";
8. Ren B. (2010). Transcription: Enhancers make non-coding RNA . Nature 465, 173-174;
9. Taganov K.D., Boldin M.P., Chang K.J., Baltimore D. (2006). NF-κB-dependent induction of miR-146 microRNA, an inhibitor targeted to signaling proteins of innate immune responses . Proc. Natl. Acad. sci. U.S.A. 103, 12481-12486;
10. O'Connell R.M., Rao D.S., Chaudhuri A.A., Boldin M.P., Taganov K.D., Nicoll J., Paquette R.L., Baltimore D. (2008). Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder. J. Exp. Med. 205, 585-594;
11. Biomolecule: "microRNA - the farther into the forest, the more firewood";
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14. Aravin A., Hannon G, Brennecke J. (2007). The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race . Science 318, 761–764;
15. Biomolecule: "

The length of siRNA is 21-25 bp; they are formed from dsRNA. The source of such RNA can be viral infections, genetic constructs introduced into the genome, long hairpins in transcripts, and bidirectional transcription of transposable elements.
dsRNAs are cut by RNase Dicer into 21–25 bp fragments. with 3" ends protruding by 2 nucleotides, after which one of the chains is part of RISC and directs the cutting of homologous RNAs. RISC contains siRNAs corresponding to both plus- and minus-strands of dsRNA. siRNAs do not have their own genes and represent are fragments of longer RNAs.siRNAs direct the cutting of the target RNA, since they are completely complementary to it.In plants, fungi, and nematodes, RNA-dependent RNA polymerases are involved in the suppression of gene expression, for which siRNAs also serve as primers (seeds for the synthesis of new RNA ) The resulting dsRNA is cut with Dicer, new siRNAs are formed, which are secondary, thus amplifying the signal.

RNA interference

In 1998, Craig C. Mello and Andrew Fire published in Nature, which stated that double-stranded RNAs (dsRNAs) were able to repress gene expression. Later it turned out that the active principle in this process is short single-stranded RNA. The mechanism of suppression of gene expression by these RNAs is called
RNA interference and RNA silencing. Such a mechanism has been found in all large taxa of eukaryotes: vertebrates and invertebrates, plants, and fungi. In 2006, the Nobel Prize was awarded for this discovery.
Suppression of expression can occur at the transcriptional level or post-transcriptionally. It turned out that in all cases a similar set of proteins and short (21-32 bp) RNAs are needed.
siRNAs regulate gene activity in two ways. As discussed above, they direct the cutting of target RNAs. This phenomenon is called "suppression" ( quelling) in mushrooms, " post-translational gene silencing"in plants and" RNA interference "in animals. siRNAs 21-23 bp long are involved in these processes. Another type of effect, siRNAs, are able to suppress the transcription of genes containing homologous siRNA sequences. This phenomenon was called transcriptional gene silencing (TGS) and found in yeast, plants and animals. siRNAs also direct DNA methylation, which leads to the formation of heterochromatin and transcriptional repression. TGS is best studied in the yeast S. pombe, where siRNAs are found to be inserted into a RISC-like protein complex called RITS. In his case, as in the case of RISC, siRNA interacts with a protein of the AGO family. Probably, siRNA is able to direct this complex to a gene that contains a homologous siRNA fragment. After that, RITS proteins recruit methyltransferases, as a result of which heterochromatin is formed in the locus encoding the siRNA target gene, and active gene expression stops.

Role in cellular processes

What is the significance of siRNA in a cell?
siRNAs are involved in cell defense against viruses, transgene repression, regulation of some genes, and formation of centromeric heterochromatin. An important function of siRNA is the suppression of the expression of mobile genetic elements. Such suppression can occur both at the transcriptional level and post-transcriptionally.
The genome of some of the viruses consists of DNA, in some of them - of RNA, moreover, RNA in viruses can be either single- or double-stranded. The process of cutting the foreign (viral) mRNA in this case occurs in the same way as described above, i.e., by activating the RISC enzyme complex. However, to be more effective, plants and insects have devised a unique way to enhance the protective effect of siRNA. By attaching to the mRNA chain, the siRNA region can, with the help of the DICER enzyme complex, first complete the second mRNA chain and then cut it in different places, thus creating a variety of "secondary" siRNAs. They, in turn, form RISC and carry mRNA through all the stages discussed above, up to its complete destruction. Such "secondary" molecules will be able to specifically bind not only to the site of the viral mRNA to which the "primary" molecule was directed, but also to other sites, which dramatically enhances the effectiveness of cellular protection.

Thus, in plants and lower animal organisms, siRNAs are an important link in a kind of "intracellular immunity" that allows them to recognize and quickly destroy foreign RNA. In the event that an RNA-containing virus enters the cell, such a protection system will prevent it from multiplying. If the virus contains DNA, the siRNA system will interfere with its production of viral proteins (since the mRNA necessary for this will be recognized and cut), and using this strategy will slow its spread throughout the body.

In mammals, unlike insects and plants, another defense system also works. When foreign RNA, the length of which is more than 30 bp, enters a "mature" (differentiated) mammalian cell, the cell begins to synthesize interferon. Interferon, binding to specific receptors on the cell surface, is able to stimulate a whole group of genes in the cell. As a result, several types of enzymes are synthesized in the cell, which inhibit the synthesis of proteins and cleave viral RNA. In addition, interferon can act on neighboring, not yet infected cells, thereby blocking the possible spread of the virus.

As you can see, both systems are similar in many ways: they have a common goal and "methods" of work. Even the names "interferon" and "(RNA) interference" themselves come from a common root. But they also have one very significant difference: if interferon, at the first signs of invasion, simply "freezes" the work of the cell, preventing (just in case) the production of many, including "innocent" proteins in the cell, then the siRNA system is extremely intelligible : each siRNA will recognize and destroy only its own specific mRNA. Substitution of just one nucleotide within siRNA leads to a sharp decrease in the effect of interference . None of the gene blockers known so far has such exceptional specificity with respect to its target gene.

The discovery of RNA interference has given new hope in the fight against AIDS and cancer. It is possible that by using siRNA therapy together with traditional antiviral therapy, a potentiation effect can be achieved, when two effects lead to a more pronounced therapeutic effect than the simple sum of each of them applied separately.
In order to use the mechanism of siRNA interference in mammalian cells, ready-made double-stranded siRNA molecules must be introduced into the cells. The optimal size of such synthetic siRNAs is the same 21–28 nucleotides. If you increase its length, the cells will respond with the production of interferon and a decrease in protein synthesis. Synthetic siRNAs can enter both infected and healthy cells, and reducing protein production in uninfected cells would be highly undesirable. On the other hand, if you try to use siRNA smaller than 21 nucleotides, the specificity of its binding to the desired mRNA and the ability to form a RISC complex sharply decrease.

If one way or another it is possible to deliver siRNA that has the ability to bind to any part of the HIV genome (which, as you know, consists of RNA), you can try to prevent it from being integrated into the DNA of the host cell. In addition, scientists are developing ways to influence the various stages of HIV reproduction in an already infected cell. The latter approach will not provide a cure, but it can significantly reduce the rate of virus reproduction and give the cornered immune system a chance to “rest” from the viral attack and try to deal with the remnants of the disease itself. In the figure, those two stages of HIV reproduction in the cell, which, as scientists hope, can be blocked using siRNA, are marked with red crosses (stages 4-5 - embedding the virus into the chromosome, and stages 5-6 - assembly of the virus and exit from the cell).


To date, however, all of the above applies only to the field of theory. In practice, siRNA therapy encounters difficulties that scientists have not been able to get around yet. For example, in the case of antiviral therapy, it is the high specificity of siRNA that can play a cruel joke: as is known, viruses have the ability to rapidly mutate, i.e. change the composition of their nucleotides. HIV has especially succeeded in this, the frequency of changes of which is such that in a person infected with one subtype of the virus, a completely different subtype can be isolated in a few years. In this case, the modified HIV strain will automatically become insensitive to the siRNA selected at the beginning of therapy.

Aging and carcinogenesis

Like any epigenetic factor, siRNAs affect the expression of genes that make them "silent". Now there are works that describe experiments to turn off genes associated with tumors. Genes are switched off (knock-down) with the help of siRNA. For example, Chinese scientists used siRNA to turn off the transcription factor 4 (TCF4) gene, the activity of which causes Pitt-Hopkins syndrome (a very rare genetic disease characterized by mental retardation and episodes of hyperventilation and apnea) and other mental illnesses. In this work, we studied the role of TCF4 in gastric cancer cells. Ectopic TCF4 expression reduces cell growth in gastric cancer cell lines, and siRNA knockout of the TCF4 gene increases cell migration. Thus, it can be concluded that epigenetic silencing of the TCF4 gene plays an important role in tumor formation and development.

According to research in the Department of Oncology, Albert Einstein Cancer Center, led by Leonard H. Augenlicht, siRNA is involved in HDAC4 gene shutdown, which causes colon cancer growth inhibition, apoptosis, and increased p21 transcription. HDAC4 is a histone deacetylase that is tissue specific, represses cell differentiation, and is downregulated during the cell differentiation process. The work shows that HDAC4 is an important regulator of colon cell proliferation (which is important in the cancer process), and it, in turn, is regulated by siRNA.

The Department of Pathology, Nara Medical University School of Medicine in Japan conducted research on prostate cancer. Replicative cell aging is a barrier against uncontrolled division and carcinogenesis. Short-lived dividing cells (TAC) are part of the prostate cell population from which the tumor develops. Japanese scientists studied the reasons why these cells overcome aging. The prostate cells in culture were transfected with junB siRNA. In these cells, an increased expression level of p53, p21, p16, and pRb is observed, which is detected during aging. Cells in culture that showed reduced levels of p16 were used for the next step. Repeated transfection of siRNA into TAC allowed the cells to avoid senescence upon p16/pRb inactivation. In addition, silencing of the junB proto-oncogene by junB siRNA causes cell invasion. Based on this, it was concluded that junB is an element for p16 and promotes cellular senescence, preventing malignancy (malignancy) of TAC. Thus, junB is a regulator of prostate carcinogenesis and may be a target for therapeutic intervention. And its activity can be regulated with the help of siRNA.

There are many such studies being carried out. At present, siRNA is not only an object, but also a tool in the hands of a medical researcher, biologist, oncologist, and gerontologist. The study of the association of siRNA with oncological diseases, with the expression of age-associated genes, is the most important task for science. Very little time has passed since the discovery of siRNA, and how many interesting studies and publications related to them have appeared. There is no doubt that their study will become one of the steps of humanity towards victory over cancer and aging...

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Small nucleolar RNAs- (snoRNA, English snoRNA) a class of small RNAs involved in chemical modifications (methylation and pseudouridylation) of ribosomal RNAs, as well as tRNAs and small nuclear RNAs. According to the MeSH classification, small nucleolar RNAs are considered a subgroup ... ... Wikipedia

small nuclear (low molecular weight nuclear) RNA- An extensive group (105 106) of small nuclear RNAs (100 300 nucleotides), associated with heterogeneous nuclear RNA, are part of the small ribonucleoprotein granules of the nucleus; M.n.RNA are a necessary component of the splicing system ... ...

small cytoplasmic RNAs- Small (100-300 nucleotides) RNA molecules localized in the cytoplasm, similar to small nuclear RNA. [Arefiev V.A., Lisovenko L.A. English Russian explanatory dictionary of genetic terms 1995 407s.] Topics genetics EN scyrpssmall cytoplasmic ... ... Technical Translator's Handbook

small nuclear RNA class U- A group of protein-associated small (from 60 to 400 nucleotides) RNA molecules that make up a significant part of the contents of the splice and are involved in the process of excising introns; in 4 out of 5 well-studied types of Usn RNA U1, U2, U4 and U5 by 5 ... ... Technical Translator's Handbook

RNA biomarkers- * RNA biomarkers * RNA biomarkers are a huge number of human transcripts that do not code for protein synthesis (nsbRNA or npcRNA). In most cases, small (miRNA, snoRNA) and long (antisense RNA, dsRNA, etc.) RNA molecules are ... ... Genetics. encyclopedic Dictionary

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