About small RNAs – for advanced dummies

About all the RNAs in the world, big and small
Peter Starokadomsky, "Biomolecule"

Twenty years ago, molecular biology did not know such an amazing phenomenon as RNA interference. Today, scientists have no doubt that this phenomenon takes part in a wide range of physiological processes in all living beings, and its molecular intermediaries – short RNA – are not inferior to blood antibodies in diversity and specificity. In protozoa, RNA interference provides immunity, in particular, protection against viruses. In more developed organisms, this mechanism is involved in the fight not only (and not so much) with external, but also with intragenomic parasites, and also becomes the most important regulator of gene activity. To date, thousands of short regulatory RNAs have already been identified, and the mechanism of RNA interference has been studied in great detail, but it is also indisputable that we are observing only the tip of this iceberg so far.

The metaphor underlying the name of the phenomenon of RNA interference refers to the experience with petunia, when the synthetase genes of pink and purple pigments artificially introduced into the plant did not increase the intensity of coloring, but, on the contrary, reduced it. Similarly, in "normal" 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 runs out, but understanding all its "twists" and deciphering the information encoded in it is truly a titanic 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 that carry information "messages" from the nucleus (with chromosomes) into the cytoplasm. The defining 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 frog oocytes by injecting the first isolated rabbit matrix RNA, encoding this protein [1].

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 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 – transport RNAs (tRNAs), which in combination with the other two – mRNA and rRNA – form a single protein-synthesizing complex. According to the rather popular hypothesis of the "world of RNA", it was this nucleic acid that lay at the very origins of life on Earth [2].

Due to the fact that RNA is significantly 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 protein synthesis begins. However, it is worth noting the "inconvenience" associated with this – RNA is very unstable. It is much worse than DNA, stored (even inside the cell) and degrades at the slightest change in conditions (temperature, pH). In addition to its "own" instability, a large contribution belongs to ribonucleases (or RNases) – a class of RNA–splitting enzymes that are 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, it is much more difficult to work with RNA than with proteins or DNA – the latter can generally be stored for hundreds of thousands of years practically without damage [3].

Fantastic accuracy at work, tridistillate, sterile gloves, disposable laboratory utensils – all this is necessary to prevent RNA degradation, but compliance with such standards has not always been possible. Therefore, for a long time, short "fragments" of RNA, inevitably polluting solutions, were simply ignored. However, over time it became clear that, despite all efforts to maintain the sterility of the working area, the "fragments" naturally continued to be detected, and then it turned out that thousands of short double-stranded RNAs are always present in the cytoplasm, performing well-defined functions, and absolutely necessary for the normal development of the cell and the organism.

The principle of RNA interferenceToday, 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:

Fig. 1. Fundamentals of RNA interference
Double-stranded RNA (dsRNA) molecules are unusual for normal cells, but they are an obligatory stage of 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 a 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 the endonuclease of the Argonaute family. Binding to kiRNA activates RISC and starts the search in the cell for DNA and RNA molecules complementary to the "template" kiRNA. The fate of such molecules is to be destroyed or inactivated by the RISC complex.Summing up, short "cuts" of foreign (including intentionally introduced) double–stranded RNA serve as a "template" for large-scale search and destruction of complementary mRNAs (and this is equivalent to suppressing the expression of the corresponding gene), and 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 protection against infections.

In 2006, Andrew Fire and Craig Mello received the Nobel Prize in Physiology or Medicine "For the 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 in general defined the regulatory mechanism of small RNAs [4] and outlined a hitherto unknown area of molecular research. Here are the main results of their work:

• With RNA interference, it is mRNA that is cleaved (and no other);
• 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;
• The dsRNA complementary to the mature mRNA site causes the 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 direction 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 in the molecular field for the "RNA interference team". Let's get to know them in more detail.

Player # 1 – Short interfering RNAsThe specificity of RNA interference is determined by short interfering RNAs (kirnas) - small double–stranded RNA molecules with a well-defined structure (see Fig.2).

Kirnas are evolutionarily the earliest, and are most widespread in plants, unicellular organisms and invertebrates [5]. In vertebrates, kirnas are practically not found in the norm, because they were replaced by later "models" of short RNAs (see below).

Kirnas – "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 3' ends and phosphorylated 5' ends. The anti-semantic mRNA is able (not by itself, of course, but with the help of the RISC complex) to recognize mRNA and specifically cause its degradation: the cut of the target mRNA always occurs exactly at the place complementary to the 10 and 11 nucleotides of the anti-semantic chain of the mRNA.

Fig. 2. The mechanism of "interference" of mRNA and kiRNA"Interfering" short RNA molecules can either enter the cell from the outside, or "cut" already in place from longer double-stranded RNAs.
The main protein required for the "slicing" of dsRNA is Dicer endonuclease. The "shutdown" of the gene by the interference mechanism is carried out by the kiRNA together with the RISC protein complex, which consists of three proteins – Ago2 endonuclease and two auxiliary proteins PACT and TRBP. Later it was discovered 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 kiRNA (the latter bypasses the "slicing" stage and immediately binds to RISC).The functions of kirnas 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, invertebrates and plants (which either do not have such a system, or it is in its infancy), the immune defense is based on RNA interference. Immunity based on RNA interference does not need complex organs to "train" the precursors of immune cells (spleen, thymus); at the same time, the variety of theoretically possible sequences of short RNAs (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 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 is no such data yet), it provides intracellular immunity more than satisfactorily.

First of all, kiRNA creates antiviral immunity by destroying the mRNA or genomic RNA of infectious organisms (for example, this is how KIRNAS were discovered in plants [6]). The introduction of viral RNA causes a powerful amplification of specific siRNAs based on the seed molecule - the viral RNA itself. In addition, siRNAs suppress the expression of various mobile genetic elements (MGE), which means that they also provide protection against endogenous "infections". Mutations in RISC-complex genes often lead to increased genome instability due to the high activity of MGE; kiRNA can be a limiter of the expression of its own genes, triggering in response to their overexpression. The regulation of genes can occur not only at the level of translation, but also during transcription – through the methylation of genes by histone H3.

In modern experimental biology, the importance of RNA interference and short RNAs cannot be overestimated. The technology of "switching off" (or knockdown) of 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, a systematic "shutdown" of all genes is carried out in turn [7].

Pharmacists have also become interested in the possibility of using kiRNA, since the ability of directed regulation of the work of individual genes promises unheard-of prospects in the treatment of a mass of diseases. The small size and high specificity of the action promise high efficiency and low toxicity of drugs based on kiRNA; however, it has not yet been possible to solve the problem of delivering kiRNA to diseased cells in the body – the reason for this is 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 at the target (inside the 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 kiRNA action can do a disservice – since viruses mutate quickly, the modified strain will very quickly lose sensitivity to the kiRNA selected at the beginning of therapy: it is known that replacing only one nucleotide in the kiRNA leads to a significant decrease in the interference effect.

At this point, it is worth recalling once again – kirnas were found only in plants, invertebrates and unicellular; although homologues of proteins for RNA interference (Dicer, RISC complex) are also present in higher animals, KIRNAS were not detected by conventional methods. What was the surprise when artificially introduced synthetic analogues of kiRNA 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 together with organisms, turning into something more "advanced". Consequently, in mammals it was necessary to look not for exact analogues of KIRNAS, but for their evolutionary successors.

Player # 2 – microRNAIndeed, based on the evolutionarily rather ancient mechanism of RNA interference, two specialized gene control systems have appeared in more developed organisms, each using its own group of small RNAs - microRNAs (microRNA) and piRNAs (piRNA, Piwi–interacting RNA).

Both systems appeared in sponges and coelenterates and evolved together with them, displacing kiRNA and the mechanism of "naked" RNA interference. Their role in providing immunity is reduced, 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 the kiRNA itself: the appearance of small double-stranded RNAs in the 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 is 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 have evolved into an additional, precise and subtle system of genome regulation. Unlike KIRNAS, 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 [8].

The microRNA precursor is transcribed from both strands of genomic DNA by RNA polymerase II, resulting in an intermediate form - pri–microRNA - bearing the signs of ordinary mRNA – m7G–cap and polyA-tail. In this precursor, a loop is formed 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 a Dicer that makes two more incisions (a double-stranded section is cut out, indicated by color in Fig. 3A). In this form, mature microRNA, similarly to kiRNA, is included in the RISC complex.

The mechanism of action of many microRNAs is similar to that of KIRNAS: 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 leads to mRNA cleavage 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 degrade, but only be reversibly blocked (there will be no translation). The same RISC complex can also use artificially introduced kirnas. This explains why kirnas 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 symmetric) organisms by combining in one figure the scheme of action of microRNAs and biotechnologically introduced siRNAs (Fig. 3B).

Rice. 3A: Structure of a double-stranded microRNA precursor molecule
Main features: the presence of conservative 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) forming a recognition site for endonucleases. The microRNA itself is highlighted in red – that's what Dicer cuts out.Fig. 3B: General mechanism of microRNA processing and realization of its activity

Fig. 3B: Generalized scheme of action of artificial microRNAs and KIRNAS

Artificial siRNAs are injected into the cell using specialized plasmids (targeting siRNA vector).microRNA functions

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 compactification, etc.). In addition, the synthesis of microRNAs themselves is regulated in a complex way (certain microRNA pools can be turned on by interferons, interleukins, tumor necrosis factor α (TNF-α) and many other cytokines). As a result, a multilevel "orchestra" tuning network of thousands of genes, stunning in its complexity and flexibility, emerges, but it does not end there either.

microRNAs are more "universal" than KIRNAS: "ward" genes do not necessarily have to be 100% complementary – regulation is carried out with 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 [9] and the development of hematopoietic stem cells [10]. The use of biochip-based technologies (micro-array screening) has shown that entire pools of small RNAs are switched on and off at various stages of cell life. Dozens of specific microRNAs have been identified for biological processes, the expression level of which varies thousands of times under certain conditions, 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 microRNAs can be radically different depending on the state of the cell! In an actively dividing cell, microRNA, binding to a complementary sequence in the 3’-region of the mRNA, 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 opposite effect – increased synthesis of the target protein [11]!

Evolution of microRNAsThe 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 about 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. For the first time, microRNAs were found in nematodes, and for a long time afterwards it was believed that these molecules appear only in sponges and coelenterates; however, they were later discovered in unicellular algae [11]. Interestingly, as organisms become more complex, the number and heterogeneity of the microRNA pool also increases. This indirectly indicates that the complexity of these organisms is provided, in particular, by the functioning of microRNAs [12]. The possible evolution of microRNAs is shown in Fig. 4.

Fig. 4. Diversity of microRNAs in different organismsThe higher the organization of the organism, the more microRNA is detected in it (the number in parentheses).
The species with single microRNAs are highlighted in red. According to [13].A clear evolutionary link can be drawn between kiRNA and microRNA based on the following facts:

• the action of both types is interchangeable and mediated by homologous proteins;
• KIRNAS injected into mammalian cells specifically "turn off" the necessary genes (despite some activation of interferon protection);
• microRNAs are being 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 forms of life based on RNA, not proteins (recall that this is the favorite theory of academician A. S. Spirin [2]).

The further, the more confusing. Player # 3 – piRNKWhile there were only two "players" in the arena of molecular biology – kiRNA 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 functionality.

However, in the process of evolution, nature has created another, evolutionarily the 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, the increase in the complexity of the genome 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 independently transcribed and migrate through the genome. Activation of such mobile elements leads to multiple DNA breaks in chromosomes, fraught with lethal consequences.

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

piRNK's "Portrait"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 sections of working genes or with fragments of the genome whose functions are unknown.

piRNAs (as well as microRNAs) are encoded in both genomic DNA chains; they are highly variable and diverse (up to 500,000 (!) species in one organism). Unlike kirnas 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 Kirnas and microRNAs, 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 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 biogenesisFigure 5A: Cytoplasmic part of piRNA processing

The biogenesis and activity of piRNA is mediated by the Piwi family of endonucleases (Ago3, Aub, Piwi). The activity of piRNA is provided by both single–stranded piRNA molecules - semantic and anti–semantic - each of which associates with a specific endonuclease Piwi. piRNA recognizes the complementary mRNA portion of the transposon (blue chain) and cuts it out. This not only inactivates the transposon, but also creates a new piRNA (bound to Ago3 by methylation with the Hen1 3’-end methylase). Such a piRNA, in turn, recognizes mRNA with transcripts of a cluster of piRNA precursors (the red chain) – in this way the cycle closes and the necessary piRNA is produced again [14].Fig. 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 to suppress the activity of MGE 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, defense against endogenous (internal) viruses is also necessary, first of all, the IGE.

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 piRNAsThe first piRNAs are found already in anemones and sponges.

Plants, apparently, went the other way – Piwi proteins were not found in them, and the role of the "muzzle" for transposons is performed by Ago4 endonuclease and kiRNA.

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 spread of piRNA in the body is so limited remains to be seen. It can be assumed that, like any powerful weapon, piRNAs benefit only in very specific conditions (during fetal development), and in an 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.

ConclusionIn 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 the simplest ones have the most developed kiRNA system (protein families Ago, Dicer), and with the complexity of organisms, the emphasis is shifted to more specialized systems - the number of protein isoforms for microRNAs increases (Drosha, Pasha) and piRNA (Piwi, Hen1).

At the same time, the diversity of enzymes mediating the action of kiRNA decreases.

Fig. 6. Diversity of proteins involved in RNA interference and
The numbers indicate the number of proteins in each group. Elements characteristic of kiRNA and microRNA are highlighted in blue, and proteins associated with piRNA are highlighted in red. According to [13].The phenomenon of RNA interference has already begun to be used by the simplest organisms.

Based on this mechanism, nature has 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 universality 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 which we are just beginning to understand.

However, such fantastic complexity rather suggests that evolution is "blind" and operates without a pre-approved "master plan" [15].

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Portal "Eternal youth" http://vechnayamolodost.ru09.06.2010