The length of siRNA is 21-25 bp, they are formed from dsRNA. The source of such RNAs can be viral infections, genetic constructs introduced into the genome, long hairpins in transcripts, and bidirectional transcription of mobile elements.
dsRNA is cut by RNase Dicer into fragments 21-25 bp long. with 3" ends protruding by 2 nucleotides, after which one of the chains is part of RISC and directs the cutting of homologous RNAs. RISC contains siRNAs corresponding to both plus and minus strands of dsRNA. siRNAs do not have their own genes and represent are fragments of longer RNAs. siRNAs direct the cutting of the target RNA, since they are completely complementary to it. In plants, fungi and nematodes, RNA-dependent RNA polymerases are involved in the process of suppressing gene expression, for which siRNAs also serve as primers (seeds for the synthesis of new RNA The resulting dsRNA is cut by Dicer, new siRNAs are formed, which are secondary, thus amplifying the signal. 
RNA interference
In 1998, Craig C. Mello and Andrew Fire published in Nature, which stated that double-stranded RNA (dsRNA) is capable of repressing gene expression. Later it turned out that the active principle in this process is short single-stranded RNA. The mechanism of suppression of gene expression using these RNAs is called
RNA interference, as well as RNA silencing. This mechanism is found in all large taxa of eukaryotes: vertebrates and invertebrates, plants and fungi. In 2006, he received the Nobel Prize for this discovery.
Suppression of expression can occur at the transcriptional level or post-transcriptionally. It turned out that in all cases a similar set of proteins and short (21-32 bp) RNAs are required.
siRNAs regulate gene activity in two ways. As mentioned above, they direct the cutting of target RNAs. This phenomenon is called "suppression" ( quelling) in mushrooms, " post-translational gene silencing"in plants and" RNA interference
"in animals. siRNAs 21-23 bp long are involved in these processes. Another type of effect is that siRNAs can suppress the transcription of genes containing homologous siRNA sequences. This phenomenon was called transcriptional gene silencing
(TGS) and is found in yeast, plants and animals. siRNAs also direct DNA methylation, which leads to heterochromatin formation and transcriptional repression. TGS is best studied in the yeast S. pombe, where siRNAs are found to be integrated into a RISC-like protein complex called RITS. In his case, as in the case of RISC, siRNA interacts with a protein of the AGO family. It is likely that siRNA is able to direct this complex to a gene that contains a homologous siRNA fragment. After this, RITS proteins recruit methyltransferases, as a result of which heterochromatin is formed in the locus encoding the siRNA target gene, and active gene expression ceases. 
Role in cellular processes
What is the significance of siRNA in a cell?
siRNAs are involved in cell protection from viruses, repression of transgenes, regulation of certain genes, and formation of centromeric heterochromatin. An important function of siRNA is suppression of the expression of mobile genetic elements. Such suppression can occur both at the transcriptional level and posttranscriptionally.
The genome of some viruses consists of DNA, while others consist of RNA, and the RNA of viruses can be either single- or double-stranded. The process of cutting foreign (viral) mRNA in this case occurs in the same way as described above, that is, by activating the RISC enzyme complex. However, for greater efficiency, plants and insects have invented a unique way to enhance the protective effect of siRNA. By joining the mRNA strand, a section of siRNA can, with the help of the DICER enzyme complex, first complete the second strand of mRNA and then cut it in different places, thus creating a variety of “secondary” siRNAs. They, in turn, form RISC and carry the mRNA through all the stages discussed above, up to its complete destruction. Such “secondary” molecules will be able to specifically bind not only to the part of the viral mRNA to which the “primary” molecule was directed, but also to other areas, which dramatically increases the effectiveness of cellular defense.
Thus, in plants and lower animal organisms, siRNAs are an important part of a kind of “intracellular immunity” that allows them to recognize and quickly destroy foreign RNA. If an RNA containing a virus has entered the cell, such a protection system will prevent it from multiplying. If the virus contains DNA, the siRNA system will prevent it from producing viral proteins (since the necessary mRNA for this will be recognized and cut), and using this strategy will slow down its spread throughout the body.
Mammals, unlike insects and plants, have a different defense system. When foreign RNA, the length of which is more than 30 bp, enters a “mature” (differentiated) mammalian cell, the cell begins to synthesize interferon. Interferon, by binding to specific receptors on the cell surface, is able to stimulate a whole group of genes in the cell. As a result, several types of enzymes are synthesized in the cell, which inhibit protein synthesis and break down viral RNA. In addition, interferon can also act on neighboring, not yet infected cells, thereby blocking the possible spread of the virus.
As you can see, both systems are similar in many ways: they have a common goal and “methods” of work. Even the names “interferon” and “(RNA) interference” themselves come from a common root. But they also have one very significant difference: if interferon, at the first signs of invasion, simply “freezes” the work of the cell, not allowing (just in case) the production of many, including “innocent” proteins in the cell, then the siRNA system is extremely intelligible : Each siRNA will recognize and destroy only its own specific mRNA. Replacement of just one nucleotide within siRNA leads to a sharp decrease in the interference effect . None of the gene blockers known so far has such exceptional specificity for its target gene.
The discovery of RNA interference has given new hope in the fight against AIDS and cancer. It is possible that by using siRNA therapy along with traditional antiviral therapy, a potentiation effect can be achieved, where the two treatments result in a greater therapeutic effect than the simple sum of each given separately.
In order to use the siRNA interference mechanism in mammalian cells, ready-made double-stranded siRNA molecules must be introduced into the cells. The optimal size of such synthetic siRNA is the same 21-28 nucleotides. If you increase its length, the cells will respond by producing interferon and reducing protein synthesis. Synthetic siRNAs can enter both infected and healthy cells, and a decrease in protein production in uninfected cells would be highly undesirable. On the other hand, if you try to use siRNA smaller than 21 nucleotides, the specificity of its binding to the desired mRNA and the ability to form the RISC complex sharply decrease.
If it is possible to deliver siRNA in one way or another that has the ability to bind to any part of the HIV genome (which, as is known, consists of RNA), one can try to prevent its integration into the DNA of the host cell. In addition, scientists are developing ways to influence various stages of HIV reproduction in an already infected cell. The latter approach will not provide a cure, but it can significantly reduce the rate of virus reproduction and give the cornered immune system a chance to “rest” from the viral attack and try to deal with the remnants of the disease itself. In the figure, the two stages of HIV reproduction in a cell, which scientists hope can be blocked using siRNA, are marked with red crosses (stages 4-5 - integration of the virus into the chromosome, and stages 5-6 - assembly of the virus and exit from the cell). 
Today, however, all of the above relates only to the field of theory. In practice, siRNA therapy encounters difficulties that scientists have not yet been able to overcome. For example, in the case of antiviral therapy, it is the high specificity of siRNA that can play a cruel joke: as is known, viruses have the ability to quickly mutate, i.e. change the composition of its nucleotides. HIV has been especially successful in this, the frequency of changes in which is such that a person infected with one subtype of the virus can, after a few years, develop a completely different subtype. In this case, the modified HIV strain will automatically become insensitive to the siRNA selected at the beginning of therapy.
Aging and carcinogenesis
Like any epigenetic factor, siRNAs affect the expression of genes that are silenced. Now there are works that describe experiments on switching off genes associated with tumors. Genes are switched off (knock-down) using siRNA. For example, Chinese scientists used siRNA to turn off the transcription factor 4 (TCF4) gene, whose activity causes Pitt-Hopkins syndrome (a very rare genetic disease characterized by mental retardation and episodes of hyperventilation and apnea) and other mental diseases. In this work, we studied the role of TCF4 in gastric cancer cells. Ectopic expression of TCF4 reduces cell growth in gastric cancer cell lines, knocking out the TCF4 gene using siRNA increases cell migration. Thus, we can conclude that epigenetic switching off (silencing) of the TCF4 gene plays an important role in the formation and development of tumors.
According to research in the Department of Oncology, Albert Einstein Cancer Center, led by Leonard H. Augenlicht, siRNA is involved in turning off the HDAC4 gene, which causes inhibition of colon cancer growth, apoptosis and increased transcription of p21. HDAC4 is a histone deacetylase that is tissue specific, inhibits cell differentiation, and its expression is suppressed during the process of cell differentiation. The work shows that HDAC4 is an important regulator of colon cell proliferation (which is important in the cancer process), and it, in turn, is regulated by siRNA.
The Department of Pathology, Nara Medical University School of Medicine in Japan conducted research on prostate cancer. Replicative cell aging is a barrier against uncontrolled division and carcinogenesis. Short-lived dividing cells (TAC) are part of the prostate cell population from which tumors form. Japanese scientists studied the reasons why these cells overcome aging. Prostate cells in culture were transfected with junB siRNA. These cells exhibit increased expression levels of p53, p21, p16 and pRb, which are detected during aging. Cells in culture that showed reduced levels of p16 were used for the next step. Repeated siRNA transfection into TAC allowed cells to avoid senescence upon p16/pRb inactivation. In addition, silencing of the junB proto-oncogene by junB siRNA causes cell invasion. Based on this, it was concluded that junB is an element for p16 and promotes cellular senescence, preventing TAC malignancy. Thus, junB is a regulator of prostate carcinogenesis and may be a target for therapeutic intervention. And its activity can be regulated using siRNA.
There are a lot of similar studies being carried out. Currently, siRNA is not only an object, but also a tool in the hands of a researcher - doctor, biologist, oncologist, gerontologist. Studying the connection between siRNA and cancer and the expression of age-associated genes is the most important task for science. Very little time has passed since the discovery of siRNA, but many interesting studies and publications related to them have appeared. There is no doubt that their study will be one of humanity’s steps towards victory over cancer and aging...
Article for the “bio/mol/text” competition: IN last years RNA - and especially its “non-classical” varieties - is attracting 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, forced us to take a closer look at RNA chemical reactions. 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 - coupled with earlier theoretical works K. Woese and, it allowed us 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 for a long time was veiled 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 is only a few percent of total length DNA, 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, it does this small RNA 6S, “imitating” 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” is logically explained a 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 environment aquatic environment(both fresh and salty), and in aquatic mollusks, and in fish, and in the human intestines - here you cannot 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 characterized 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 amount 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.
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), preventing the translation of mRNA on ribosomes into the protein it encodes. Ultimately, the effect of small interfering RNA is identical to that of simply reducing gene expression.
Small interfering RNAs were discovered in 1999 by David Baulcombe's group in the UK as a component of a post-transcriptional gene silencing system in plants. PTGS, en:post-transcriptional gene silencing). The team published their findings in the journal Science.
Double-stranded RNA can enhance gene expression through a mechanism called RNA-dependent gene activation. RNAa, small RNA-induced gene activation). It has been shown that double-stranded RNAs complementary to the promoters of target genes cause activation of the corresponding genes. RNA-dependent activation upon administration of synthetic double-stranded RNA has been demonstrated for human cells. It is not known whether a similar system exists in the cells of other organisms.
By providing the ability to turn off essentially any gene at will, small interfering RNA-based RNA interference has generated enormous interest in basic and applied biology. The number of broad-based RNAi-based tests to identify important genes in biochemical pathways is growing. Since the development of diseases is also determined by the activity of genes, it is expected that in some cases, switching off a gene using small interfering RNA may have a therapeutic effect.
However, the application of small interfering RNA-based RNA interference to animals, and especially to humans, faces many difficulties. Experiments have shown that the effectiveness of small interfering RNA is different for different types cells: some cells readily respond to small interfering RNA and demonstrate a decrease in gene expression, while in others this is not observed, despite effective transfection. The reasons for this phenomenon are still poorly understood.
Results from phase 1 trials of the first two RNAi therapeutics (intended to treat macular degeneration), published in late 2005, show that small interfering RNA drugs are easily tolerated by patients and have acceptable pharmacokinetic properties.
Preliminary clinical trials of small interfering RNAs targeting the Ebola virus indicate that they may be effective for post-exposure prophylaxis of the disease. This drug allowed the entire group of experimental primates to survive after receiving a lethal dose of the Zaire Ebolavirus
Destruction of the target mRNA can also occur under the influence of small interfering RNA (siRNA). RNA interference is one of the new revolutionary discoveries in molecular biology, and its authors received a Nobel Prize. Interfering RNAs are very different in structure from other types of RNA and are two complementary RNA molecules approximately 21-28 nitrogen bases long, which are connected to each other like strands in a DNA molecule. In this case, two unpaired nucleotides always remain at the edges of each siRNA chain. The impact is carried out as follows. When a siRNA molecule finds itself inside a cell, at the first stage it binds into a complex with two intracellular enzymes - helicase and nuclease. This complex was called RISC ( R NA- i induced s ilencing c complex; silence - English be silent, shut up; silencing - silencing, this is how the process of “turning off” a gene is called in English and specialized literature). Next, the helicase unwinds and separates the siRNA strands, and one of the strands (antisense in structure) in complex with the nuclease specifically interacts with the complementary (strictly corresponding to it) region of the target mRNA, which allows the nuclease to cut it into two parts. The cut sections of mRNA are then exposed to the action of other cellular RNA nucleases, which further cut them into smaller pieces.
SiRNAs found in plants and lower animal organisms (insects) are an important part of a kind of “intracellular immunity” that allows them to recognize and quickly destroy foreign RNA. If an RNA containing a virus has entered the cell, such a protection system will prevent it from multiplying. If the virus contains DNA, the siRNA system will prevent it from producing viral proteins (since the necessary mRNA for this will be recognized and cut), and using this strategy will slow down its spread throughout the body. It has been established that the siRNA system is extremely discriminating: each siRNA will recognize and destroy only its own specific mRNA. Replacement of just one nucleotide within siRNA leads to a sharp decrease in the interference effect. None of the gene blockers known so far has such exceptional specificity for its target gene.
Currently, this method is mainly used in scientific research to identify the functions of various cellular proteins. However, it could potentially also be used to create medicines.
The discovery of RNA interference has given new hope in the fight against AIDS and cancer. It is possible that by using siRNA therapy in conjunction with traditional antiviral and anticancer therapies, a potentiation effect can be achieved, where the two treatments result in a greater therapeutic effect than the simple sum of each given alone.
In order to use the siRNA interference mechanism in mammalian cells for therapeutic purposes, ready-made double-stranded siRNA molecules must be introduced into the cells. However, there are a number of problems that currently do not allow this to be done in practice, much less to create any dosage forms. Firstly, in the blood they are affected by the first echelon of the body’s defense, enzymes - nucleases, which cut potentially dangerous and unusual double strands of RNA for our body. Secondly, despite their name, small RNAs are still quite long, and, most importantly, they carry a negative electrostatic charge, which makes their passive penetration into the cell impossible. And thirdly, one of the most important questions is how to make siRNA work (or penetrate) only in certain (“sick”) cells, without affecting healthy ones? And finally there is the issue of size. The optimal size of such synthetic siRNA is the same 21-28 nucleotides. If you increase its length, the cells will respond by producing interferon and reducing protein synthesis. On the other hand, if you try to use siRNA smaller than 21 nucleotides, the specificity of its binding to the desired mRNA and the ability to form the RISC complex sharply decrease. It should be noted that overcoming these problems is critical not only for siRNA therapy, but also for gene therapy in general.
Some progress has already been made in solving them. For example, scientists are trying to chemical modifications make siRNA molecules more lipophilic, that is, capable of dissolving in the fats that make up the cell membrane, and thus facilitating the penetration of siRNA into the cell. And in order to ensure specificity of work within only certain tissues, genetic engineers include in their constructs special regulatory sections, which are activated and trigger the reading of the information contained in such a construct (and therefore siRNA, if it is included there), only in certain cells fabrics.
So, researchers from the University of California, San Diego School of Medicine have developed a new effective system for delivering small interfering RNA (siRNA), which suppresses the production of certain proteins, into cells. This system should become the basis of technology for specific delivery of drugs to Various types cancerous tumors. “Small interfering RNAs, which carry out a process called RNA interference, have incredible potential for treating cancer,” explains Professor Steven Dowdy, who led the research: “and although we still have a lot of work to do, this moment“We have developed a technology for delivering drugs to a population of cells – both the primary tumor and metastases, without damaging healthy cells.”
For many years, Dowdy and his colleagues have been studying the anticancer potential of small interfering RNAs. However, conventional siRNAs are tiny, negatively charged molecules that, due to their properties, are extremely difficult to deliver into cells. To achieve this, scientists used a short signaling protein PTD (peptide transduction domain). Previously, more than 50 “hybrid proteins” were created with its use, in which PTD was combined with tumor suppressor proteins.
However, simply connecting siRNA to PTD does not lead to delivery of RNA into the cell: siRNA is negatively charged, PTD is positively charged, resulting in the formation of a dense RNA-protein conglomerate that is not transported across the cell membrane. So the researchers first coupled the PTD to a protein RNA-binding domain that neutralized the negative charge of the siRNA (resulting in a fusion protein called PTD-DRBD). Such an RNA-protein complex easily passes through the cell membrane and enters the cell cytoplasm, where it specifically inhibits the messenger RNA proteins that activate tumor growth.
To test the ability of the PTD-DRBD fusion protein to deliver siRNA into cells, the scientists used a cell line derived from human lung cancer. After treating cells with PTD-DRBD-siRNA, it was found that tumor cells were most susceptible to siRNA, while in normal cells (T cells, endothelial cells and embryonic stem cells were used as controls), where there was no increased production of oncogenic proteins, no toxic effects were observed.
This method can be subjected to various modifications using different siRNAs to suppress different tumor proteins - not only those produced in excess, but also mutant ones. It is also possible to modify therapy in case of relapse of tumors, which usually become resistant to chemotherapy drugs due to new mutations.
Oncological diseases are very variable, and the molecular characteristics of tumor cell proteins are individual for each patient. The authors of the work believe that in this situation, the use of small interfering RNA is the most rational approach to therapy.
Scientists believe that incorrect expression of small RNAs is one of the causes of a number of diseases that seriously affect the health of many people around the world. These diseases include cardiovascular 23 and cancer 24 . As for the latter, this is not surprising: cancer indicates anomalies in the development of cells and their fate, and small RNAs play vital role in the relevant processes. Here is one of the very illustrative examples the enormous impact that small RNAs have on the body during cancer. It's about about a malignant tumor, which is characterized by incorrect expression of those genes that act during the initial development of the organism, and not in the postnatal period. This is a type of childhood brain tumor that usually appears before the age of two. Unfortunately, this is a very aggressive form of cancer, and the prognosis here is unfavorable even with intensive treatment. The oncological process develops due to improper redistribution genetic material in brain cells. A promoter that normally drives strong expression of one of the protein-coding genes undergoes recombination with a specific cluster of small RNAs. Then this entire rearranged region undergoes amplification: in other words, many copies of it are created in the genome. Consequently, small RNAs located “downstream” of the relocated promoter are expressed much more strongly than they should be. The level of active small RNAs is approximately 150-1000 times higher than normal.
Rice. 18.3. Small RNAs activated by alcohol can combine with messenger RNAs that do not affect the body's resistance to the effects of alcohol. But these small RNAs do not bind to the messenger RNA molecules that promote such resistance. This results in a relative predominance of the proportion of messenger RNA molecules encoding protein variations associated with alcohol tolerance.
This cluster encodes more than 40 different small RNAs. In fact, this is generally the largest of such clusters found in primates. It is usually expressed only at an early stage human development, in the first 8 weeks of embryonic life. Its strong activation in the infant brain leads to catastrophic effects on genetic expression. One consequence is the expression of an epigenetic protein that adds modifications to the DNA. This leads to widespread changes in the entire pattern of DNA methylation, and therefore to abnormal expression of all sorts of genes, many of which should only be expressed when immature brain cells divide during the early stages of development. This is how the cancer program starts in the baby's cells 25.
Such communication between small RNAs and the epigenetic machinery of the cell can have a significant impact on other situations when cells develop a predisposition to cancer. This mechanism likely results in the effect of disruption of small RNA expression being enhanced by changes in epigenetic modifications that are transmitted to daughter cells from the mother. This can create a pattern of potentially dangerous changes in the pattern of gene expression.
So far, scientists have not figured out all the stages of the interaction of small RNAs with epigenetic processes, but they can still get some hints about the features of what is happening. For example, it turned out that a certain class of small RNAs, which enhance the aggressiveness of breast cancer, targets certain enzymes in messenger RNAs that remove key epigenetic modifications. This alters the pattern of epigenetic modifications in the cancer cell and further disrupts genetic expression 26 .
Many forms of cancer are difficult to track in a patient. Oncological processes can occur in hard-to-reach places, which complicates the sampling procedure. In such cases, it is not easy for the doctor to monitor the development of the cancer process and the response to treatment. Often doctors are forced to rely on indirect measurements - say, a tomographic scan of a tumor. Some researchers believe that small RNA molecules could help create a new technique for monitoring tumor development, which could also study its origin. When cancer cells die, small RNAs leave the cell when it ruptures. These small junk molecules often form complexes with cellular proteins or are wrapped in fragments cell membranes. Due to this, they are very stable in body fluids, which means that such RNAs can be isolated and analyzed. Since their quantities are small, researchers will have to use very sensitive analysis methods. However, nothing is impossible here: the sensitivity of nucleic acid sequencing is constantly increasing 27 . Data have been published confirming the promise of this approach in relation to breast cancer 28 , ovarian cancer 29 and a number of other oncological diseases. Analysis of small circulating RNAs in lung cancer patients has shown that these RNAs help distinguish between patients with a solitary pulmonary nodule (not requiring therapy) and patients who develop malignant tumor nodules (requiring treatment) 30 .
