Small interfering RNAs. See what “Small RNA” is in other dictionaries Functions of small RNA

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

In a living cell, the flow of information between the nucleus and the cytoplasm never dries up, but understanding all its “swirls” and deciphering the information encoded in it is truly a Herculean task. One of the most important breakthroughs in biology of the last century can be considered the discovery of information (or matrix) RNA (mRNA or mRNA) molecules, which serve as intermediaries carrying information “messages” from the nucleus (from chromosomes) to the cytoplasm. The decisive role of RNA in protein synthesis was predicted back in 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 rabbit messenger RNA encoding this protein for the first time.

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 not template, 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 RNAs (tRNAs), which in combination with two others - mRNA and rRNA - form a single protein-synthesizing complex. According to the fairly popular “RNA world” hypothesis, 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 with ribose), it is more labile and can move relatively freely in the cell, and therefore deliver short-lived replicas of genetic information (mRNA) to the place where it begins protein synthesis. However, it is worth noting the “inconvenience” associated with this - RNA is very unstable. It is much worse stored than DNA (even inside a cell) and degrades at the slightest change in conditions (temperature, pH). In addition to the “own” instability, a large contribution belongs to ribonucleases (or RNases) - a class of RNA-cleaving enzymes that are very stable and “ubiquitous” - even the skin of the experimenter’s hands contains enough of these enzymes to negate 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 virtually no damage.

Fantastic care during work, tridistillate, sterile gloves, disposable laboratory glassware - all this is necessary to prevent RNA degradation, but maintaining such standards was not always possible. Therefore, for a long time, they simply did not pay attention to short “fragments” of RNA, which inevitably contaminated solutions. However, over time, it became clear that, despite all efforts to maintain the sterility of the work area, “debris” naturally continued to be discovered, and then it turned out that thousands of short double-stranded RNAs are always present in the cytoplasm, performing very specific functions, and are absolutely necessary for normal development cells and organism.

Principle of RNA interference

Today, the study of small regulatory RNAs is one of the most rapidly developing areas of molecular biology. It was discovered that all short RNAs perform their functions based on 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). The mechanism of RNA interference is shown very schematically in Fig. 1:

Rice. 1. Basics of RNA interference
Double-stranded RNA (dsRNA) molecules are uncommon in normal cells, but they are an essential step in the life cycle of many viruses. A special protein called Dicer, having detected dsRNA in the cell, “cuts” it into small fragments. The antisense strand of such a fragment, which can already be called short interfering RNA (siRNA, from siRNA - small interference RNA), is bound by a complex of proteins 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 in the cell for DNA and RNA molecules that are complementary to the “template” siRNA. The fate of such molecules is to be destroyed or inactivated by the RISC complex.

To summarize, short “cuts” of foreign (including intentionally introduced) double-stranded RNA serve as a “template” for a large-scale search and destruction of complementary mRNA (and this is equivalent to suppression of the expression of the corresponding gene), not only in one cell, but also in neighboring ones. For many organisms - protozoa, mollusks, 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 had been 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 a hitherto unknown area of molecular research. Here are the main results of their work:

During RNA interference, it is the mRNA (and no other) that is cleaved;
Double-stranded RNA acts (causes cleavage) much more efficiently than single-stranded RNA. 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 an entire area of modern molecular biology - RNA interference - and determined the vector of work of many research groups around the world for decades. To date, three large groups of small RNAs have been discovered that play on the molecular field as the “RNA interference team.” Let's get to know them in more detail.

Player #1 – short interfering RNA

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

siRNAs are the earliest in evolution and are most widespread in plants, single-celled organisms and invertebrates. In vertebrates, practically no siRNAs are normally found, because they were replaced by later “models” of short RNAs (see below).

siRNA - “templates” for searching in the cytoplasm and destroying mRNA molecules - have a length of 20–25 nucleotides and a “special feature”: 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 the RISC complex) to recognize mRNA and specifically cause its degradation: the target mRNA is cut at the exact site complementary to the 10th and 11th nucleotides of the anti-sense siRNA chain.

Rice. 2. Mechanism of “interference” between mRNA and siRNA
“Interfering” short RNA molecules can either enter the cell from the outside or be “cut” in place from longer double-stranded RNA. The main protein required for cutting dsRNA is the 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 – the endonuclease Ago2 and two auxiliary proteins PACT and TRBP. Later it was discovered that the Dicer and RISC complexes can use as a “seed” not only dsRNA, but also single-stranded RNA that forms 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 thing 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 based on RNA interference. Immunity based on RNA interference does not require complex “training” organs for immune cell precursors (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 of higher animals. In addition, siRNAs are synthesized on the basis of the “hostile” RNA that has infected the cell, which means, unlike antibodies, they are immediately “tailored” for a specific type of infection. And although protection based on RNA interference does not work outside the cell (at least, there is no such data yet), it provides intracellular immunity more than satisfactorily.

First of all, siRNA creates antiviral immunity by destroying mRNA or genomic RNA of infectious organisms (for example, this is how siRNAs were discovered in plants). The introduction of viral RNA causes powerful amplification of specific siRNAs based on the primer molecule - the viral RNA itself. In addition, siRNAs suppress the expression of various mobile genetic elements (MGEs), and therefore 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 act as a limiter on the expression of its own genes, triggering in response to their overexpression. Regulation of gene function can occur not only at the level of translation, but also during transcription - through methylation of genes at histone H3.

In modern experimental biology, the importance of RNA interference and short RNAs can hardly be overestimated. A technology has been developed to “turn off” (or knockdown) individual genes in vitro (on cell cultures) and in vivo (on embryos), which has already become a de facto standard when studying 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 specifically regulate the functioning of individual genes promises unprecedented prospects in the treatment of a host of diseases. 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 delivering siRNA to diseased cells in the body - this is due to the fragility and fragility of these molecules. And although dozens of teams are now trying to find a way to direct these “magic bullets” exactly to the target (inside diseased organs), they have not yet achieved visible success. Besides this, 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 mutate quickly, the modified strain will very quickly lose sensitivity to the siRNA selected at the beginning of therapy: it is known that replacing just one nucleotide in siRNA leads to a significant decrease interference effect.

At this point it is worth recalling once again - siRNAs were found only in plants, invertebrates and unicellular organisms; Although homologues of proteins for RNA interference (Dicer, RISC complex) are also present in higher animals, siRNAs were not 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 the organisms, turning into something more “advanced.” Consequently, in mammals it was necessary to look not for exact analogues of siRNAs, but for their evolutionary successors.

Player #2 – microRNA

Indeed, based on the evolutionarily ancient mechanism of RNA interference, more developed organisms have developed two specialized systems for controlling the operation of genes, each using its own group of small RNAs - microRNA and 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 decreasing, 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 triggers the siRNA itself: the appearance of small double-stranded RNA 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 microRNAs and piRNAs - single-stranded molecules with a specific structure that are not detected by the interferon system.

As the genome became more complex, microRNAs and piRNAs became increasingly involved in the regulation of transcription and translation. Over time, they turned into an additional, precise and subtle system of genome regulation. Unlike siRNA, microRNA and piRNA (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 the appearance of an intermediate form—pri-microRNA—carrying the features of regular mRNA—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, the endonuclease Drosha 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, who makes two more cuts (a double-stranded section is cut out , color coded in Fig. 3A). In this form, the mature microRNA, similar to siRNA, is included in the RISC complex.

The mechanism of action of many microRNAs is similar to the action of siRNAs: a short (21–25 nucleotides) single-stranded RNA as part of the RISC protein complex binds with high specificity to the complementary site in the 3’-untranslated region of the target mRNA. Binding leads to the cleavage of the mRNA by the Ago protein. However, the activity of microRNA (compared to siRNA) 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 complement the illustration of the mechanism of action of RNA interference in higher (bilaterally symmetric) organisms by combining in one figure the action diagram of microRNAs and biotechnologically introduced siRNAs (Fig. 3B).

Rice. 3A: 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; a specific sequence (2–8 bp) that forms a recognition site for endonucleases. The microRNA itself is highlighted in red—it is what Dicer cuts out.

Rice. 3B: General mechanism of microRNA processing and implementation of its activity

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

Functions of microRNA

The physiological functions of microRNAs are extremely diverse - in fact, they act as the main non-protein regulators of ontogenesis. microRNAs do not cancel, but complement the “classical” scheme of gene regulation (inducers, suppressors, chromatin compaction, etc.). In addition, the synthesis of microRNAs themselves is complexly regulated (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 tuning an “orchestra” of thousands of genes emerges, amazing in its complexity and flexibility, but this does not end there.

microRNAs are more “universal” than siRNAs: “ward” genes do not have to be 100% complementary - regulation is also carried out through partial interaction. Today, one of the hottest topics in molecular biology is the search for microRNAs that act as alternative regulators of known physiological processes. For example, microRNAs involved in the regulation of the cell cycle and apoptosis in plants, Drosophila and nematodes have already been described; in humans, microRNAs regulate the immune system and the development of hematopoietic stem cells. The use of biochip-based technologies (micro-array screening) has shown that entire pools of small RNAs are switched on and off at different stages of cell life. Dozens of specific microRNAs have been identified for biological processes, 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 – completely or partially – the work of genes. However, it recently turned out that the action of microRNAs can differ radically depending on the state of the cell! In an actively dividing cell, microRNA binds to a complementary sequence in the 3' region of the mRNA and inhibits protein synthesis (translation). However, in a state of rest or stress (for example, when growing in a poor environment), the same event leads to the exact opposite effect - increased synthesis of the target protein!

Evolution of microRNA

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 say there are 700 microRNAs, and this number is constantly growing). microRNAs regulate the activity of about 30% of all genes (the 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 prevalence of microRNAs indicate an evolutionarily ancient origin. MicroRNAs were first discovered in nematodes, and for a long time it was believed that these molecules appear only in sponges and coelenterates; however, they were later discovered in unicellular algae. Interestingly, as organisms become more complex, the number and heterogeneity of the miRNA pool also increases. This indirectly indicates that the complexity of these organisms is provided, in particular, by the functioning of microRNAs. The possible evolution of miRNAs is shown in Fig. 4.

Rice. 4. Diversity of microRNAs in different organisms
The higher the organization of the organism, the more microRNAs are found in it (the number in parentheses). Species in which single microRNAs were found are highlighted in red. According to .

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

the action of both types is interchangeable and is mediated by homologous proteins;
siRNAs introduced into mammalian cells specifically “turn off” the desired genes (despite some activation of interferon protection);
microRNAs are being discovered 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 forms of life based on RNA, and 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 carries out similar actions; As organisms become more complex, so does 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 is (or vice versa? ;-). However, increasing genome complexity also has a downside: a 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 be autonomously transcribed and migrate throughout the genome. Activation of such transposable elements leads to multiple DNA breaks in chromosomes, which can have lethal consequences.

The number of MGEs increases nonlinearly with genome size, and their activity must be contained. To do this, animals, starting with coelenterates, use the same phenomenon of RNA interference. This function is also performed by short RNAs, but not by the ones already discussed, but by a 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 chain with a characteristic feature - uracil (U) at the 5' end and a methylated 3' end. There are other differences:

Unlike siRNAs and microRNAs, they do not require processing by Dicer;
piRNA genes are active only in germ cells (during embryogenesis) and the surrounding endothelial cells;
The protein composition of the piRNA system is different - these are endonucleases of the Piwi class (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
The biogenesis and activity of piRNAs is mediated by the Piwi family of endonucleases (Ago3, Aub, Piwi). The activity of piRNA is provided by both single-stranded piRNA molecules - sense and anti-sense - each of which associates with a specific Piwi endonuclease. The 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 methylation of the 3' end by Hen1 methylase). This piRNA, in turn, recognizes mRNA with transcripts from the piRNA precursor cluster (red strand) - in this way the cycle is closed and the desired piRNA is produced again.

Rice. 5B: piRNA in the nucleus
In addition to the Aub endonuclease, the 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.

Functions of piRNA

The main function of piRNA is to suppress MGE activity at the level of transcription and translation. It is believed that piRNAs are active only during embryogenesis, when unpredictable genome shuffling is especially dangerous and can lead to the death of the embryo. This is logical - when the immune system has not yet started working, the cells of the embryo need some simple but effective protection. The embryo is reliably protected from external pathogens by the placenta (or egg shell). But in addition to this, defense is also necessary 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 disruption of the development of germ cells.

Distribution and evolution of piRNAs

The first piRNAs are already found in sea anemones and sponges. Plants apparently took a different path - 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, piRNAs are beneficial only under very specific conditions (during fetal development), and in the adult body their activity will cause more harm than good. Still, the number of piRNAs exceeds the number of known proteins by an order of magnitude, and the nonspecific effects of piRNAs in mature cells are difficult to predict.

Pivot table. Properties of all three classes of short RNAs

	siRNA	microRNA	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-chain complex structure	Single-chain 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 translation of target mRNA	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 provide 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 as organisms become more complex, 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 that mediate the action of siRNA decreases.

Rice. 6. Diversity 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 RNAs (see summary table) - as a result, we see thousands of fine 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 only beginning to understand.

True, such fantastic complexity rather suggests that evolution is “blind” and acts 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: “MicroRNAs discovered for the first time in a single-celled 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 microRNA miR-146, 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 further into the forest, the more firewood”;
Elements: “The complication of the body 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: "

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

RNA: more than just a copy of DNA

Most readers of this site have known the basic mechanisms of a living cell since school. In biology courses, from Mendel's laws to cutting-edge genome sequencing projects, the red thread runs through the idea of a major genetic program for the development of an organism, known to professional biologists as central dogma of molecular biology. It states that the DNA molecule acts as a carrier and keeper of genetic information, which, through an intermediary - messenger RNA (mRNA), and with the participation of ribosomal (rRNA) and transfer RNA (tRNA), - is realized in the form of proteins. The latter determine the species and individual phenotype.

This state of affairs and the assignment of RNA to the role of a minor participant in the molecular performance persisted in the scientific community until the 80s of the last century. The work of T. Chek, who showed that RNA can act as a catalyst for chemical reactions, forced us to take a closer look at RNA. Previously, it was believed that the acceleration of chemical processes in a cell is the prerogative of enzymes that are exclusively protein in nature. The discovery of catalytic activity in RNA had far-reaching consequences - together with the earlier theoretical works of K. Woese and, it made it possible to draw a possible picture of prebiotic evolution on our planet. The fact is that since the discovery of DNA's function as a carrier of genetic information, the dilemma of what appeared earlier in the course of evolution - DNA or the protein necessary for the reproduction of DNA - seemed almost as philosophical (that is, pointless) as the question about the primacy of the appearance of the chicken or the egg. After the discovery of T. Chek, the solution took on very real shape - a molecule was found that had the properties of both an information carrier and a biocatalyst (albeit in its rudimentary form). Over time, these studies grew into a whole direction in biology, studying the origin of life through the prism of the so-called “RNA world”.

So it became obvious that the ancient world of RNA could be related to the origin and flourishing of primary life. However, it does not automatically follow from this that RNA in modern organisms is not an archaism adapted to the needs of intracellular molecular systems, but a truly important participant in the molecular ensemble of the cell. Only the development of molecular methods - in particular, nucleic acid sequencing - showed that RNA is truly irreplaceable in the cell, and not only in the form of the canonical trinity “mRNA, rRNA, tRNA”. Already the first extensive data on DNA sequencing pointed to a fact that at first seemed difficult to explain - most of it turned out to be non-coding- that is, not carrying information about protein molecules or “standard” RNA. Of course, this can be partially attributed to “genetic debris” - “switched off” or lost function genome fragments. But saving such an amount of “dowry” for biological systems that try to spend energy sparingly seems illogical.

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

Small RNAs are a Trojan horse for bacterial proteins

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

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

RNA molecules that must bind to proteins to perform their function.
RNAs that bind complementarily to other RNAs (comprise the majority of known RNA regulatory molecules).

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

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

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

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

The mechanism by which regulators of the second group operate is, in general, similar to that of regulatory RNAs in eukaryotes - this is the same RNA interference through hybridization with mRNA, only the chains of small RNAs themselves are often longer - up to several hundred nucleotides ( cm. rice. 1). As a result, due to small RNA, ribosomes cannot read information from mRNA. Although often, it seems, it does not come to this: the resulting “small RNA - mRNA” complexes become the target of RNases (such as RNase P).

The compactness and packing density of the prokaryotic genome makes itself felt: if in eukaryotes most regulatory RNAs are written in separate (most often not protein-coding) loci, then many small RNAs of bacteria can be encoded in the same DNA region as the suppressed gene, but on the opposite chains! These RNAs are called cis-encoded(antisense), and small RNAs lying at some distance from the suppressed section of DNA - trans-encoded. Apparently, the arrangement of cis-RNAs can be considered a triumph of ergonomics: they can be read from the opposite DNA strand at the moment of its unwinding simultaneously with the target transcript, which makes it possible to finely control the amount of protein synthesized.

Small RNAs in trans evolve independently of the target mRNA, and the sequence of the regulator changes more strongly as a result of mutations. Perhaps this arrangement is only beneficial for the bacterial cell, since small RNA acquires activity against previously unusual targets, which reduces the time and energy costs for creating other regulators. On the other hand, selection pressure prevents trans-small RNA from mutating too much because it will lose activity. However, to hybridize with messenger RNA, most trans-small RNAs require a helper, the Hfq protein. Apparently, otherwise, incomplete complementarity of the small RNA may create problems for binding to the target.

Apparently, the potential regulatory mechanism based on the principle of “one small RNA - many targets” helps to integrate the metabolic networks of the bacterium, which is extremely necessary in conditions of a short single-cell life. One can continue speculating on the topic and assume that with the help of trans-encoded small RNAs, expression “instructions” are sent from functionally related, but physically distant loci. The need for this kind of genetic “roll call” logically explains the large number of small RNAs found in pathogenic bacteria. For example, several hundred small RNAs were found in the record holder for this indicator - Vibrio cholerae ( Vibrio cholerae). This is a microorganism that can survive in the surrounding aquatic environment (both fresh and salty), and on aquatic shellfish, and in fish, and in the human intestines - there is no way to do without complex adaptation with the help of regulatory molecules!

CRISPR protects bacterial health

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

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

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

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

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

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

Riboswitch - sensor for bacteria

So, there are protein-associating small RNAs, there are small RNAs that interfere with the bacteria’s own mRNA, and also RNAs captured by bacteria from viruses and suppressing phage DNA. Is it possible to imagine any other mechanism of regulation using small RNAs? It turns out yes. If we analyze what was described above, we will find that in all cases of antisense regulation, interference of small RNA and the target is observed as a result of hybridization of two individual molecules. Why not arrange small RNA as part of the transcript itself? Then it is possible, by changing the conformation of such a “misplaced Cossack” inside the mRNA, to change the accessibility of the entire template for reading during translation or, which is even more energetically expedient, to regulate the biosynthesis of mRNA, i.e. transcription!

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

The described schematic diagram of the riboswitch device is not canon; there are variations. A curious “turn-on” tandem riboswitch was discovered in Vibrio cholerae: the expression platform is preceded by two at once aptamer region. Obviously, this provides greater sensitivity and a smoother response to the appearance of another amino acid in the cell - glycine. Perhaps, a “double” riboswitch in the genome of the anthrax pathogen, similar in principle of action, is indirectly involved in the high survival rate of the bacterium ( Bacillus anthracis). It reacts to a compound that is part of the minimal medium and is vital for this microbe - thiamine pyrophosphate.

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

No need for proteins, give us RNA!

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

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

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

It will be interesting to see how our understanding of small RNA regulators will change when new data are obtained on RNAseq platforms, including on free-living and uncultured forms. Recent work using “deep sequencing” has already yielded unexpected results, indicating the presence of microRNA-like molecules in mutant streptococci. Of course, such data need careful double-checking, but be that as it may, we can confidently say that the study of small RNAs in bacteria will bring many surprises.

Acknowledgments

The original ideas and compositional design when creating the title picture, as well as picture 4, belong to a graduate of the Institute of Archiology of the Southern Federal University Kopaeva E.A. The presence of Figure 2 in the article is the merit of the associate professor of the department. Zoology SFU G.B. Bakhtadze. He also carried out scientific proofreading and revision of the title figure and Figure 4. The author expresses his deep gratitude to them for their patience and creative approach to the matter. Special thanks to my colleague, senior researcher. lab. biochemistry of microbes of the Rostov Anti-Plague Institute Sorokin V.M. for discussing the text of the article and making valuable comments.

Literature

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

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 truly a Herculean task. One of the most important breakthroughs in biology of the last century can be considered the discovery of information (or matrix) RNA (mRNA or mRNA) molecules, which serve as intermediaries carrying information “messages” from the nucleus (from chromosomes) to the cytoplasm. The decisive role of RNA in protein synthesis was predicted back in 1939 in the work of Thorbjörn Kaspersson ( Torbjörn Caspersson), Jean Bracheta ( Jean Brachet) and Jack Schultz ( Jack Schultz), and in 1971 George Marbeis ( George Marbaix) triggered hemoglobin synthesis 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 not template, 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 RNAs (tRNAs), which in combination with two others - mRNA and rRNA - form a single protein-synthesizing complex. According to the fairly popular “RNA world” hypothesis, it was this nucleic acid that lay at the very origins of life on Earth.

Fantastic care during work, tri-distillate, sterile gloves, disposable laboratory glassware - all this is necessary to prevent RNA degradation, but maintaining such standards was not always possible. Therefore, for a long time, they simply did not pay attention to short “fragments” of RNA, which inevitably contaminated solutions. However, over time, it became clear that, despite all efforts to maintain the sterility of the work area, “debris” naturally continued to be discovered, and then it turned out that thousands of short double-stranded RNAs are always present in the cytoplasm, performing very specific functions, and are absolutely necessary for normal development cells and organism.

Principle of RNA interference

Pharmacists have also become interested in the possibility of using siRNA, since the ability to specifically regulate the functioning of individual genes promises unprecedented prospects in the treatment of a host of diseases. 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 been successful - this is due to the fragility and fragility of these molecules. And although dozens of teams are now trying to find a way to direct these “magic bullets” exactly to the target (inside diseased organs), they have not yet achieved visible success. Besides this, 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 replacing just one nucleotide in siRNA leads to a significant decrease interference effect.

At this point it is worth recalling again - siRNAs were discovered only in plants, invertebrates and unicellular organisms; Although homologues of proteins for RNA interference (Dicer, RISC complex) are also present in higher animals, siRNAs were not 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 the organisms, turning into something more “advanced.” Consequently, in mammals it was necessary to look not for exact analogues of siRNAs, but for their evolutionary successors.

Player #2 - microRNA

Indeed, based on the evolutionarily quite ancient mechanism of RNA interference, two specialized systems for controlling the operation of genes appeared in more developed organisms, 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 decreasing, 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 triggers the siRNA itself: the appearance of small double-stranded RNA 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 microRNAs and piRNAs - single-stranded molecules with a specific structure that are not detected by the interferon system.

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

Meet: microRNA

The microRNA precursor is transcribed from both strands of genomic DNA by RNA polymerase II, resulting in the appearance of an intermediate form - pri-microRNA - which carries the features of ordinary 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 two-stage processing (Fig. 4): first, the endonuclease Drosha cuts off single-stranded RNA “tails” from the hairpin, after which the excised hairpin (pre-microRNA) is exported to the cytoplasm, where it is recognized by Dicer, who makes two more cuts (a double-stranded section is cut out , indicated by color in Fig. 3). In this form, the mature microRNA, similar 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; a specific sequence (2–8 bp) that forms a recognition site for endonucleases. The microRNA itself is highlighted in red - this is what Dicer cuts out.

The mechanism of action of many microRNAs is similar to the action of siRNAs: a short (21–25 nucleotides) single-stranded RNA as part of the RISC protein complex binds with high specificity to the complementary site in the 3′ untranslated region of the target mRNA. Binding leads to the cleavage of the mRNA by the Ago protein. However, the activity of microRNA (compared to siRNA) 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 be used artificially introduced siRNA. This explains why siRNAs made by analogy with protozoa are also active in mammals.

Figure 5. Generalized scheme of action of artificial microRNAs and siRNAs(artificial siRNAs are introduced into the cell using specialized plasmids - targeting siRNA vector).

Functions of microRNA

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

Evolution of microRNA

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 say there are 700 microRNAs, and this number is constantly growing). microRNAs regulate the activity of about 30% of all genes (the 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.

Figure 6. MicroRNA diversity in different organisms. The higher the organization of the organism, the more microRNAs are found in it (the number in parentheses). Species in which they were found are highlighted in red. single microRNA.

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

the action of both types is interchangeable and is mediated by homologous proteins;
siRNAs introduced into mammalian cells specifically “turn off” the desired genes (despite some activation of interferon protection);
microRNAs are being discovered in more and more ancient organisms.

The further you go, the more confusing it becomes. Player #3 - piRNA

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 carries out similar actions; As organisms become more complex, so does functionality.

The more complex the genome is organized, the more developed and adapted the organism is (or vice versa? ;-). However, the increase in genome complexity also has a downside: a 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 ( MGE) - one of the main factors of genome instability - are short unstable regions that can be autonomously transcribed and migrate throughout the genome. Activation of such transposable elements leads to multiple DNA breaks in chromosomes, which can have lethal consequences.

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

“Portrait” of piRNA

Functions of piRNA

Distribution and evolution of piRNAs

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, piRNAs are beneficial only under very specific conditions (during fetal development), and in the adult body their activity will cause 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	microRNA	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-chain complex structure	Single-chain complex structure. U at 5′ end, 2′ end 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 translation of target mRNA	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 provide 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 as organisms become more complex, 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 that mediate the action of siRNA decreases.

Figure 9. Diversity of proteins involved in RNA interference(numbers indicate the number of proteins of each group). Blue elements characteristic of siRNA and microRNA are highlighted, and red- protein And piRNA-related.

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 RNAs ( cm. tab. 1) - as a result, we see thousands of fine 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 only beginning to understand.

(True, such fantastic complexity rather suggests that evolution is “blind” and acts 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;