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

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

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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 there are always thousands of short double-stranded RNAs in the cytoplasm that perform quite specific functions and are 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 as a seed, but also single-stranded RNA forming a double-stranded hairpin, as well as ready-made siRNA (the latter bypasses the “cutting” stage and immediately binds to RISC).

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 miRNAs, they are formed in a single strand with a characteristic 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: "

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 there are always thousands of short double-stranded RNAs in the cytoplasm that perform quite specific functions and are 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 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 - 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 microRNAs 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

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 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;

), preventing translation of mRNA on ribosomes into the protein it encodes. Ultimately, the effect of small interfering RNAs is identical to that of simply reducing gene expression.

Small interfering RNAs were discovered in 1999 by David Balcombe's group in the UK as a component of the post-transcriptional gene silencing system in plants (eng. PTGS, post-transcriptional gene silencing). The group published their findings in the journal Science.

Double-stranded RNAs can increase gene expression through a mechanism called RNA-dependent gene activation. RNAa, small RNA-induced gene activation). It has been shown that double-stranded RNAs complementary to promoters of target genes cause activation of the corresponding genes. RNA-dependent activation upon administration of synthetic double-stranded RNAs has been shown in human cells. It is not known whether a similar system exists in the cells of other organisms.

With the ability to turn off essentially any gene at will, RNA interference based on small interfering RNAs has generated tremendous interest in basic and applied biology. The number of wide-ranging RNAi-based assays to identify important genes in biochemical pathways is constantly growing. Since the development of diseases is also determined by the activity of genes, it is expected that in some cases, turning off a gene using small interfering RNA can have a therapeutic effect.

However, the application of RNA interference based on small interfering RNAs to animals, and in particular to humans, faces many difficulties. Experiments have shown that the effectiveness of small interfering RNAs is different for different types of cells: some cells easily respond to the action of small interfering RNAs and show a decrease in gene expression, while in others this is not observed, despite effective transfection. The reasons for this phenomenon are still poorly understood.

The results of the first phase trials of the first two RNAi therapeutic drugs (intended for the treatment of macular degeneration), published at the end of 2005, show that drugs based on small interfering RNAs are easily tolerated by patients and have acceptable pharmacokinetic properties.

Preliminary clinical trials of small interfering RNAs targeting Ebola virus indicate that they may be effective for post-exposure prophylaxis of the disease. This drug allowed the survival of the entire group of experimental primates who received a lethal dose of Zairean Ebolavirus.