About all the RNA in the world, big and small. Big things for small molecules: how small RNAs orchestrate bacterial genes The spread and evolution of piRNAs

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 .

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.

Pivot table. Properties of all three classes of short RNAs

	siRNA	miRNA	piRNA
Spreading	Plants, Drosophila, C.elegans. Not found in vertebrates	eukaryotes	Embryonic cells of animals (starting with coelenterates). Not in protozoa and plants
Length	21-22 nucleotides	19-25 nucleotides	24-30 nucleotides
Structure	Double-stranded, 19 complementary nucleotides and two unpaired nucleotides at the 3' end	Single strand complex structure	Single-stranded complex structure. U at 5'-end, 2'- O-methylated 3' end
Processing	Dicer-dependent	Dicer-dependent	Dicer-independent
Endonucleases	Ago2	Ago1, Ago2	Ago3, Piwi, Aub
Activity	Degradation of complementary mRNAs, acetylation of genomic DNA	Degradation or inhibition of target mRNA translation	Degradation of mRNA encoding MGE, regulation of MGE transcription
Biological role	Antiviral immune defense, suppression of the activity of one's own genes	Regulation of gene activity	Suppression of MGE activity during embryogenesis

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

Gurdon J.B., Lane C.D., Woodland H.R., Marbaix G. (1971). Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells . Nature 233, 177-182;
Spirin A. S. (2001). Protein Biosynthesis, the RNA World and the Origin of Life. Bulletin of the Russian Academy of Sciences 71, 320-328;
Elements: "Complete mitochondrial genomes of extinct animals can now be extracted from hair";
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;
Biomolecule: "MicroRNA found for the first time in a unicellular organism";
Covey S., Al-Kaff N., Lángara A., Turner D. (1997). Plants combat infection by gene silencing. Nature 385, 781-782;
Biomolecule: "Molecular Double Dealing: Human Genes Work for the Influenza Virus";
Ren B. (2010). Transcription: Enhancers make non-coding RNA . Nature 465, 173-174;
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;
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;
Biomolecule: "microRNA - the farther into the forest, the more firewood";
Elements: "The complication of the organism in ancient animals was associated with the emergence of new regulatory molecules";
Grimson A., Srivastava M., Fahey B., Woodcroft B.J., Chiang H.R., King N., Degnan B.M., Rokhsar D.S., Bartel D.P. (2008). Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature 455, 1193-1197.
Aravin A., Hannon G, Brennecke J. (2007). The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race . Science 318, 761–764;
Biomolecule: "

Small hairpin RNAs or short hairpin RNAs (shRNA short hairpin RNA, small hairpin RNA) are short RNA molecules that form dense hairpins in the secondary structure. ShRNA can be used to turn off expression ... ... Wikipedia

RNA polymerase- from a T. aquaticus cell in the process of replication. Some elements of the enzyme are made transparent, and the RNA and DNA chains are more clearly visible. Magnesium ion (yellow) is located on the active site of the enzyme. RNA polymerase is an enzyme that carries out ... ... Wikipedia

RNA interference- Delivery of small hairpin RNAs using a lentivirus vector and the mechanism of RNA interference in mammalian cells RNA interference (a ... Wikipedia

RNA gene Non-coding RNA (ncRNA) are RNA molecules that are not translated into proteins. The previously used synonym, small RNA (smRNA, small RNA), is currently not used, since some non-coding RNAs can be very ... ... Wikipedia

Small nuclear RNA- (snRNA, snRNA) a class of RNAs that are found in the nucleus of eukaryotic cells. They are transcribed by RNA polymerase II or RNA polymerase III and are involved in important processes such as splicing (removal of introns from immature mRNA), regulation ... Wikipedia

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

Books

Buy for 1877 UAH (Ukraine only)
Clinical genetics. Textbook (+CD), Bochkov Nikolai Pavlovich, Puzyrev Valery Pavlovich, Smirnikhina Svetlana Anatolyevna. All chapters have been revised and supplemented in connection with the development of medical science and practice. The chapters on multifactorial diseases, prevention, treatment of hereditary diseases,…

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 Thorbjorn Kaspersson ( Torbjorn Caspersson), Jean Brachet ( Jean Brachet) and Jack Schultz ( Jack Schultz), and in 1971 by George Marbeis ( George Marbaix) triggered the synthesis of hemoglobin in frog oocytes by injecting the first isolated rabbit messenger RNA encoding this protein.

In 1956–1957, 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 was described and studied - transfer RNA (tRNA), 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 "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 a sufficient amount 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, tridisstylate, 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

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 solve the problem delivery siRNA to diseased cells in the body has not yet succeeded - the reason for this is 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 siRNA action can be a disservice - since viruses quickly mutate, the modified strain will very quickly lose sensitivity to siRNA selected at the beginning of therapy: it is known that replacing just one nucleotide in siRNA leads to a significant decrease in interference effect.

At this point, it is worth recalling once again - siRNAs were found only in plants, invertebrates and unicellular; 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 produced 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 quite 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 - miRNA(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, miRNAs and piRNAs (discovered in 2001, see Box 3) are not produced from foreign double-stranded RNA molecules, but are initially encoded in the host's genome.

Meet microRNA

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 - m 7 G-cap and polyA tail. This precursor forms a loop with two single-stranded “tails” and several unpaired nucleotides in the center (Fig. 3). Such a loop undergoes a two-stage processing (Fig. 4): first, the 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. 3). In this form, mature miRNA, similarly to siRNA, is included in the RISC complex.

Figure 3. Structure of a double-stranded microRNA 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 microRNA itself is highlighted in red - that is what Dicer cuts out.

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 siRNA. This explains why siRNAs, made by analogy with protozoa, are also active in mammals.

Thus, we can complete 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 microRNAs and biotechnologically introduced siRNAs (Fig. 5).

Figure 5. 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

microRNAs 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 microRNAs 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, miRNA 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

Figure 6. 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 are marked in red in which single miRNA.

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.

The further, the more confusing. Player #3 - piRNA

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 ( SHP) - one of the main factors of genome instability - are short unstable regions that can be autonomously transcribed and migrate through the genome. Activation of such transposable elements leads to multiple DNA breaks in chromosomes, which are fraught with lethal consequences.

"Portrait" of piRNA

piRNA functions

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

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 the nonspecific effects of piRNAs in mature cells are difficult to predict.

Table 1. Properties of all three classes of short RNAs

	siRNA	miRNA	piRNA
Spreading	Plants, Drosophila, C.elegans. Not found in vertebrates	eukaryotes	Embryonic cells of animals (starting with coelenterates). Not in protozoa and plants
Length	21–22 nucleotides	19–25 nucleotides	24–30 nucleotides
Structure	Double-stranded, 19 complementary nucleotides and two unpaired nucleotides at the 3' end	Single strand complex structure	Single-stranded complex structure. U at the 5'-end, 2'- O-methylated 3′ end
Processing	Dicer-dependent	Dicer-dependent	Dicer-independent
Endonucleases	Ago2	Ago1, Ago2	Ago3, Piwi, Aub
Activity	Degradation of complementary mRNAs, acetylation of genomic DNA	Degradation or inhibition of target mRNA translation	Degradation of mRNA encoding MGE, regulation of MGE transcription
Biological role	Antiviral immune defense, suppression of the activity of one's own genes	Regulation of gene activity	Suppression of MGE activity during embryogenesis

Conclusion

In conclusion, I would like to give a table illustrating the evolution of the protein apparatus involved in RNA interference (Fig. 9). 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.

Figure 9. Variety of proteins involved in RNA interference(numbers indicate the number of proteins in each group). in blue elements characteristic of siRNA and microRNA are highlighted, and red- protein and associated with piRNA.

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 kinds of short RNAs ( cm. tab. 1) - 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 that evolution is “blind” and operates without a pre-approved “master plan”»;

Andrew Grimson, Mansi Srivastava, Bryony Fahey, Ben J. Woodcroft, H. Rosaria Chiang, et. al. (2008). Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature. 455 , 1193-1197;

A. A. Aravin, G. J. Hannon, J. Brennecke. (2007). The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race . Science. 318 , 761-764;

A.M. Deichman, S.V. Zinoviev, A.Yu. Baryshnikov

GENE EXPRESSION AND SMALL RNA IN ONCOLOGY

GU RONTS im. N.N.Blokhina RAMS, Moscow

SUMMARY

The article presents the role of small RNAs that control most of the vital functions of the cell and the body, and their possible connection, in particular, with oncogenesis and other (including hypothetical) intracellular mechanisms of genomic expression.

Keywords Key words: small RNAs, RNA interference (RNAi), double-stranded RNA (lncRNA), RNA editing, oncogenesis.

A.M. Deichman, S.V.Zinoviev, A.Yu.Baryshnikov.

THE GENE EXPRESSION AND SMALL RNAS IN ONCOLOGY

N.N. Blokhin Russian Cancer Research Center RAMS, Moscowow

ABSTRACT

In the paper role of small RNAs supervising the majority vital functions of cell and organism and possible connection of them in particular with oncogenesis and others (including hypothetical) intracellular mechanisms of genome expression is submitted.

key words: Small RNAs, interference RNAs (RNAi), double strand RNAs (dsRNAs), RNA editing, tumorogenesis.

Introduction

The expression of individual genes and entire eukaryotic genomes, including processing, various types of transcription, splicing, rearrangements, RNA editing, recombinations, translation, RNA interference, is regulated by some proteins (products of regulatory, structural, homeotic genes, transcription factors), mobile elements, RNA and low molecular weight effectors. Processing RNAs include rRNA, tRNA, mRNA, some regulatory RNAs, and small RNAs.

To date, it is known that small RNAs do not code for protein, often number in the hundreds per genome, and are involved in the regulation of the expression of various eukaryotic genes (somatic, immune, germline, stem cells). Under control are the processes of differentiation, (hematopoiesis, angiogenesis, adipogenesis, myogenesis, neurogenesis), morphogenesis (including embryonic stages, development/growth, physiological regulation), proliferation, apoptosis, carcinogenesis, mutagenesis, immunogenesis, aging (life extension), epigenetic silencing ; cases of metabolic regulation (eg, glycosphingolipids) have been noted. A wider class of non-coding RNAs of 20-300/500 nucleotides and their RNPs were found not only in the nucleus/nucleolus/cytoplasm, but also in DNA-containing cell organelles (animal mitochondria; in plants, micro-RNAs and sequences of small RNA).

For the management and regulation of V.N. processes it is important: 1. that small natural/artificial RNAs (small RNAs, tRNAs, etc.) and their complexes with proteins (RNPs) are capable of transmembrane cellular and mitochondrial transport; 2. that after the collapse of mitochondria, part of their contents, RNA and RNP, may end up in the cytoplasm and nucleus. The listed properties of small RNAs (RNPs), whose functionally significant role is only increasing in the process of study, obviously have a connection with the factor of alertness in relation to cancer and other genetic diseases. At the same time, the high significance of epigenomic modifications of chromatin in the development of tumors became clear. We will consider only a very limited number of cases out of many similar ones.

Small RNA

The mechanism of action of small RNAs is their ability to bind almost complementarily to 3'-untranslated regions (3'-UTRs) of target mRNAs (which sometimes contain DNA/RNA transposing MIR/LINE-2 elements, as well as conserved Alu repeats). ) and induce RNA interference (RNAi=RNAi; particularly in an antiviral response). The complication, however, is that, in addition to cellular ones, there are also virus-encoded small RNAs (herpes, SV40, etc.; EBV, for example, contains 23, and KSHV - 12 miRNAs), interacting with the transcripts of both the virus and the host. More than 5,000 cellular/viral miRNAs alone are known in 58 species. RNAi initiates either degradation (with the participation of the RISC complex, RNA-Induced Silencing Complex) at nuclease-vulnerable fragments of continuous helixes of lncRNA (double-stranded RNA mRNA, etc.), or partially reversible inhibition of discontinuously coiled lncRNA during translation of mRNA targets. Mature small RNAs (~15-28 nucleotides) are formed in the cytoplasm from their precursors of various lengths (tens and hundreds of nucleotides) processing in the nucleus. In addition, small RNAs are involved in the formation of the silencing structure of chromatin, the regulation of transcription of individual genes, the suppression of transposon expression, and the maintenance of the functional structure of extended sections of heterochromatin.

There are several main types of small RNAs. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are the most well studied. In addition, among small RNAs, the following are studied: piRNAs active in germline cells; small interfering RNAs associated with endogenous retrotransposons and repeating elements (with local/global heterochromatization - starting from the early stages of embryogenesis; maintain the level of telomeres), Drosophia rasiRNAs; often encoded by introns of protein genes and functionally important for translation, transcription, splicing (de-/methylation, pseudouridylation of nucleic acids) small nuclear (snRNAs) and nucleolar (snoRNAs) RNAs; complementary to DNA-binding NRSE-(Neuron Restrictive Silenser Element) motifs small modulatory RNAs, smRNAs, with little-known functions; transactivating plant small interfering RNAs, tasiRNAs; short hairpin RNAs, shRNAs, providing long-term RNAi (persistent gene silencing) of long lncRNA structures in the antiviral response in animals.

Small RNAs (miRNAs, siRNAs, etc.) interact with newly synthesized transcripts of the nucleus/cytoplasm (regulating splicing, translation of mRNA; methylation/pseudouridylation of rRNA, etc.) and chromatin (with temporally local and epigenetically inherited heterochromatinization of dividing somatic germ cells). Heterochromatinization, in particular, is accompanied by de-/methylation of DNA, as well as methylation, acetylation, phosphorylation and ubiquitination of histones (modification of the "histone code").

The miRNAs of the nematode Caenorhabditis elegans (lin-4), their properties and genes were the first to be discovered and studied among small RNAs, and somewhat later, the miRNAs of the plant Arabidopsis thaliana. Currently, they are associated with multicellular organisms, although they are shown in the unicellular alga Chlamydomonas reinhardtii, and RNAi-like silencing pathways, in connection with antiviral / similar protection involving the so-called. psiRNAs are discussed for prokaryotes. The genomes of many eukaryotes (including Drosophila and humans) contain several hundred miRNAs genes. These stage/tissue-specific genes (as well as their corresponding target mRNA regions) are often highly homologous in phylogenetically distant species, but some of them are lineage-specific. miRNAs are contained in exons (protein-coding, RNA genes), introns (most often pre-mRNA), intergenic spacers (including repeats), have a length of up to 70-120 nucleotides (or more) and form loop/stem hairpin structures. To determine their genes, not only biochemical and genetic, but also computer approaches are used.

The most characteristic length of the "working region" of mature miRNAs is 21-22 nucleotides. These are perhaps the most numerous of the non-protein-coding genes. They can be arranged as separate copies (more often) or clusters containing many similar or different miRNAs genes transcribed (not rarely from autonomous promoters) as a longer precursor, processed in several stages to individual miRNAs. It is assumed that there is a regulatory miRNA network that controls many fundamental biological processes (including oncogenesis/metastasis); probably at least 30% of human expressed genes are regulated by miRNAs.

This process involves lncRNA-specific RNase-III-like enzymes Drosha (nuclear ribonuclease; initiates the processing of intron pre-miRNAs after splicing of the main transcript) and Dicer, which functions in the cytoplasm and cleaves/degrades, respectively, hairpin pre-miRNAs (to mature miRNAs). ) and later hybrid miRNAs/mRNA structures. Small RNAs, together with several proteins (including h.p. RNases, proteins of the AGO family, transmethylases / acetylases, etc.) and with the participation of the so-called. RISC- and RITS-like complexes (the second one induces transcriptional silencing) are capable, respectively, of inducing RNAi/degradation and subsequent gene silencing at the RNA- (before/during translation) and DNA- (during transcription of heterochromatin) levels.

Each miRNA potentially pairs with multiple targets, and each target is controlled by a number of miRNAs (similar to gRNAs-mediated pre-mRNA editing in trypanosome kinetoplasts). In vitro analysis showed that miRNAs regulation (as well as RNA editing) is a key posttranscriptional modulator of gene expression. Similar miRNAs competing for the same target are potential transregulators of RNA-RNA and RNA-protein interactions.

In animals, miRNAs are best studied for the nematode Caenorhabditis Elegans; over 112 genes have been described. Thousands of endogenous siRNAs have also been found here (there are no genes; they are associated, in particular, with spermatogenesis-mediated transcripts and transposons). Both small multicellular RNAs can be generated by RNA polymerases that exhibit activity (not homology) of RdRP-II (as for most other RNAs) and RdRP-III types. Mature small RNAs are similar in composition (including terminal 5'-phosphates and 3'-OH), length (typically 21-22 nucleotides), and function, and may compete for the same target. However, RNA degradation, even when the target is fully complementary, is more often associated with siRNAs; translational repression, with partial, usually 5-6 nucleotides, complementarity with miRNAs; and the precursors, respectively, are exo-/endogenous (hundreds/thousands of nucleotides) for siRNAs, and usually endogenous (tens/hundreds of nucleotides) for miRNAs, and their biogenesis is different; however, in some systems these differences are reversible.

RNAi mediated by siRNAs- and miRNAs has a variety of natural roles: from regulation of gene expression and heterochromatin to protection of the genome against transposons and viruses; but siRNAs and some miRNAs are not conserved between species. Plants (Arabidopsis thaliana) have: siRNAs corresponding to both genes and intergenic (including spacers, repeats) regions; a huge number of potential genome sites for various types of small RNAs. Nematodes also have so-called. variable autonomously expressed 21U-RNAs (dasRNAs); they have 5 "-Y-monophosphate, make up 21 nucleotides (20 of them are variable), and are located between or inside the introns of protein-coding genes at more than 5700 sites in two regions of chromosome IV.

MiRNAs play an important role in gene expression in health and disease; a person has at least 450-500 such genes. By binding usually to the 3 "-UTR regions of mRNA (other targets), they can selectively and quantitatively (in particular, when removing products of low-expressed genes from circulation), block the work of some and the activity of other genes. It turned out that the sets of profiles of expressed micro- RNAs (and their targets) dynamically change during ontogenesis, differentiation of cells and tissues.These changes are specific, in particular, during cardiogenesis, the process of optimizing the size of the length of dendrites and the number of synapses of a nerve cell (with the participation of miRNA-134, other small RNAs), the development of many pathologies (oncogenesis, immunodeficiencies, genetic diseases, parkinsonism, Alzheimer's disease, ophthalmic disorders (retinoblastoma, etc.) associated with infections of various nature) The total number of detected miRNAs is growing much faster than the description of their regulatory role and association with specific targets .

Computer analysis predicts hundreds of target mRNAs for individual miRNAs and regulation of individual mRNAs by multiple miRNAs. Thus, miRNAs can serve the purpose of eliminating target gene transcripts or fine-tuning their expression at the transcriptional/translational levels. Theoretical considerations and experimental results support the existence of diverse roles for miRNAs.

A more complete list of aspects related to the fundamental role of small RNAs in eukaryotes in growth/development processes and in some pathologies (including cancer epigenomics) is reflected in the review.

Small RNAs in Oncology

The processes of growth, development, progression and metastasis of tumors are accompanied by many epigenetic changes that develop into more rare persistently inherited genetic changes. Rare mutations, however, can carry a lot of weight (for a specific individual, nosology), because. in relation to individual genes (for example, APC, K-ras, p53), the so-called. the “funnel” effect associated with almost irreversible development/consequences of oncological diseases. Tumor-specific in relation to the expression profile of various genes (proteins, RNA, small RNA) progenitor cell heterogeneity is due to coupled variations of rearranged epigenomic structures. The epigenome is modulated by methylation, post-translational modifications/substitutions of histones (with non-canonical ones), remodeling of the nucleosomal structure of genes/chromatin (including genomic imprinting, i.e., dysfunction in the expression of alleles of parental genes and X chromosomes). All this, and with the participation of RNAi regulated by small RNAs, leads to the appearance of defective heterochromatic (including hypomethylated centromeric) structures.

The formation of gene-specific mutations may be preceded by the known accumulation of hundreds of thousands of somatic clonal mutations in simple repeats or microsatellites of the non-coding (rarely coding) region, at least in tumors with a microsatellite mutator phenotype (MMP); they make up a significant part of colorectal, as well as cancers of the lung, stomach, endometrium, etc. Unstable mono-/heteronucleotide microsatellite repeats (poly-A6-10, similar) are found many times more often in regulatory non-coding genes that control the expression of genes (introns, intergenic) than in the coding (exons) regions of the genome of microsatellite-unstable, MSI+, tumors. Although the nature of the appearance and mechanisms of localization of MS-stable/unstable regions are not completely clear, the formation of MS-instability correlated with the frequency of mutations of many genes that did not mutate earlier in MSI+ tumors and probably channeled the pathways of their progression; moreover, the mutation rate of MSI repeats in these tumors increased by more than two orders of magnitude. Not all genes have been analyzed for the presence of repeats, but the degree of their mutability in coding/noncoding regions is different, and the accuracy of methods for determining the frequency of mutations is relative. It is important that non-coding regions for MSI-mutable repeats are often biallelic, while coding regions are monoallelic.

A global decrease in methylation in tumors is characteristic of repeats, transposable elements (MEs; their transcription increases), promoters, CpG sites of tumor suppressor miRNA genes, and correlates with hypertranscription of retrotransposons in advanced cancer cells. Normally, “methylome” fluctuations are associated with parental/stage/tissue-specific “methylation waves” and strong methylation of centromeric satellite regions of heterochromatin regulated by small RNAs. When satellites are undermethylated, the formed chromosome instability is accompanied by an increase in recombination, and a violation of TE methylation can trigger their expression. These factors favor the development of the tumor phenotype. Small RNA therapy can be highly specific but should be controlled because targets can be not only individual, but also many mRNA / RNA molecules, and newly synthesized RNA of various (including non-coding intergenic repeats) regions of chromosomes.

Most of the human genome is made up of repeats and TEs. Retrotransposon L1 (LINE element) contains, like endogenous retroviruses, reversetase (RTase), endonuclease and is potentially capable of carrying non-autonomous (Alu, SVA, etc.) retroelements; silencing of L1/similar elements occurs as a result of methylation at CpG sites. Note that among the CpG sites of the genome, CpG islands of gene promoters are weakly methylated, and 5-methylcytosine itself is a potentially mutagenic base that is deaminated into thymine (chemically, or with the participation of RNA/(DNA) editing, DNA repair); however, some of the CpG islands are subject to excessive aberrant methylation accompanied by suppressor gene repression and cancer development. Next: RNA-binding protein encoded by L1, interacting with proteins AGO2 (of the Argo-naute family) and FMRP (fragile mental retardation, effector RISC-complex protein), promotes the movement of the L1 element - which indicates a possible mutual regulation of systems RNAi and retropositions of human LINE elements. It is important, in particular, that Alu repeats are able to move into the region of the intron/exon space of genes.

These and similar mechanisms can enhance the pathological plasticity of the tumor cell genome. Suppression of RTase (encoded, like endonuclease, by L1 elements; RTase is also encoded by endogenous retroviruses) by the RNAi mechanism was accompanied by a decrease in proliferation and an increase in differentiation in a number of cancer cell lines. Upon introduction of the L1 element into a proto-oncogene or a suppressor gene, DNA double-strand breaks were observed. In tissues of the germline (mice/human), the level of L1 expression is increased, and its methylation depends on the piRNAs-(26-30-bp)-associated silencing system, where PIWI proteins are variants of the large family of Argo-naute proteins, mutations in which lead to demethylation/derepression of L1/like elements with long terminal repeats. The silencing pathways of rasiRNAs are associated with PIWI proteins to a greater extent than with Dicer-1/2 and Ago proteins. The piRNAs/siRNAs-mediated silencing pathways are realized through intranuclear bodies containing large evolutionarily conserved multiprotein PcG complexes, whose functions are often impaired in tumor cells. These complexes are responsible for long-range action (through more than 10 kb, between chromosomes) and regulate the cluster of HOX genes responsible for body plan.

New principles of antisense therapy can be developed taking into account knowledge of more highly specific (than histone-modifying inhibitors of DNA/protein methylation) antitumor epigenomic agents, the fundamental principles of epigenomic RNA silencing, and the role of small RNAs in carcinogenesis.

Micro-RNA in Oncology

It is known that an increase in tumor growth and metastasis can be accompanied by an increase in some and a decrease in the expression of other individual/sets of miRNAs (Table 1). Some of them may have a causative role in oncogenesis; and even the same miRNAs (like miR-21/-24) in different tumor cells can exhibit both oncogenic and suppressive properties. Each type of human malignant tumors is clearly distinguishable by its "miRNA-imprint", and some miRNAs can function as oncogenes, tumor suppressors, initiators of cell migration, invasion, metastasis. In pathologically altered tissues, a reduced number of key miRNAs, probably included in the anti-cancer defense systems, is often found. miRNAs (miRs) involved in oncogenesis have formed the concept of the so-called. "oncomirax": analysis of the expression of more than 200 miRNAs from over 1000 samples of lymphomas and solid cancers has successfully classified tumors into subtypes according to their origin and stage of differentiation. The functions and role of miRNAs are successfully studied using: anti-miR oligonucleotides modified (to increase the lifetime) at 2'-O-methyl and 2'-O-methoxyethyl groups; as well as LNA oligonucleotides, in which the oxygen atoms of ribose in positions 2 "and 4" are connected by a methylene bridge.

(Table 1)……………….

Tumor	miRNAs
Lungs' cancer	17-92 , let-7↓ , 124a↓ , 126 ↓ , 143 ↓ , 145 ↓ , 155 , 191 , 205 , 210
Mammary cancer	21 , 125b↓ , 145 ↓ , 155
Prostate cancer	15a ↓ , 16-1 ↓ , 21 , 143 ↓ ,145 ↓
bowel cancer	19a , 21 , 143 ↓ , 145 ↓
Pancreas cancer	21 , 103 , 107 , 155 v
ovarian cancer	210
Chronic lymphocytic leukemia	15a ↓ , 16-1 ↓ , 16-2 , 23 b , 24-1 , 29 ↓ , 146 , 155 , 195 , 221 , 223 ↓

Table 1 .

miRNAs whose expression increases () or decreases ( ↓ ) in some of the more common tumors compared to normal tissues (see also ).

It is believed that the regulatory role of expression, disappearance, and amplification of miRNA genes in the predisposition to initiation, growth, and progression of most tumors is significant, and mutations in miRNA/mRNA-target pairs are synchronized. The expression profile of miRNAs can be used for classification, diagnosis, and clinical prognosis in oncology. Changes in the expression of miRNAs may affect the cell cycle, the cell's survival program. Mutations of miRNAs in stem and somatic cells (as well as selection of polymorphic target mRNA variants) may contribute to, or even play a critical role in, the growth, progression, and pathophysiology of many (if not all) malignant neoplasms. With the help of miRNAs, apoptosis correction is possible.

In addition to individual miRNAs, their clusters were found, acting as an oncogene that provokes the development, in particular, of hematopoietic tissue cancer in experimental mice; miRNAs genes with oncogenic and suppressor properties can be located in the same cluster. Cluster analysis of miRNAs expression profiles in tumors makes it possible to determine its origin (epithelium, hematopoietic tissue, etc.) and classify different tumors of the same tissue with non-identical transformation mechanisms. miRNAs expression profiling can be performed using nano-/microarrays; the accuracy of such a classification, when the technology is developed (which is not easy), turns out to be higher than with the use of mRNA profiles. Some of the miRNAs are involved in the differentiation of hematopoietic cells (mouse, human), initiating the progression of cancer cells. Human miRNA genes are often located in the so-called. "fragile" sites, areas with a predominance of deletions / insertions, point breaks, translocations, transpositions, minimally deletable and amplified heterochromatin regions involved in oncogenesis.

Angiogenesis . The role of miRNAs in angiogenesis is probably significant. An increase in angiogenesis in some Myc-activated human adenocarcinomas was accompanied by a change in the expression pattern of some miRNAs, while gene knockdown of other miRNAs led to a weakening and suppression of tumor growth. Tumor growth was accompanied by mutations in K-ras, Myc and TP53 genes, increased production of angiogenic VEGF factor and the degree of Myc-associated vascularization; while the antiangiogenic factors Tsp1 and CTGF were suppressed by miR-17-92 and other cluster-associated miRNAs. Tumor angiogenesis and vascularization were enhanced (particularly in colonocytes) when two oncogenes were co-expressed to a greater extent than one.

Neutralization of the anti-angiogenic factor LATS2, an inhibitor of animal cyclin-dependent kinase (CDK2; human/mouse), with miRNAs-372/373 ("potential oncogenes") stimulated testicular tumor growth without damaging the p53 gene.

Potential modulators of angiogenic properties (in-vitro/in-vivo) are miR-221/222, whose targets, c-Kit receptors (others), are angiogenesis factors of umbilical cord endothelial venous HUVEC cells, etc. These miRNAs and c-Kit interact as part of a complex cycle that controls the ability of endothelial cells to form new capillaries.

Chronic lymphocytic leukemia (CLL). In B-cell chronic lymphocytic leukemia (CLL), a reduced level of expression of the miR-15a/miR-16-1 genes (and others) is noted in the 13q14 region of the human chromosome - the site of the most common structural anomalies (including deletions of the 30kb region), although the genome expressed hundreds of mature and pre-human miRNAs. Both miRNAs potentially effective in tumor therapy contained antisense regions of the anti-apoptotic Bcl2 protein, suppressed its overexpression, stimulated apoptosis, but were almost/completely absent in two-thirds of the stray CLL cells. Frequent mutations of sequenced miRNAs in stem/somatic cells were identified in 11 of 75 patients (14.7%) with a family predisposition to CLL (mode of inheritance unknown), but not in 160 healthy patients. These observations raise the suggestion of a direct function of miRNAs in leukemogenesis. Currently, not everything is known about the relationship between miRNAs gene expression levels (and their functions) and other genes in normal/tumor cells.

Document

Relevance. Violation of the function of the facial nerve during surgery on the parotid salivary gland is one of the urgent problems and is determined both by the prevalence of the disease and by a significant frequency.

Dawson Church - the genius in your genes epigenetic medicine and the new biology of intention www e - puzzle ru library book www e - puzzle ru library table of contents

Book

Ethics spirituality oncology hiv p garyaev* a enfi summary

Document

This article reflects a new view on the problem of oncology and HIV infection in the light of Linguistic-Wave Genetics (LVG) and Essence Coding Theory (ESC) on the basis of Russian and other socio-cultural realities.

Oncology Research Center and Anastasia Sergeevna Odintsova new regimens of chemotherapy for advanced and recurrent cervical cancer 14 01 12 – oncology

Thesis

4.4. Determination of the uridinglucoronyltransferase isoenzyme gene (UGT1A1) in the blood serum of patients with cervical cancer who received first-line chemotherapy with irinotecan with platinum derivatives 105