RNA interference
The RNA interference ( RNAi for short or RNA silencing ) is a natural mechanism in the cells of living organisms with a nucleus ( eukaryotes ), which is used for targeted silencing genes. It is a special case of gene silencing. RNA interference, based on an interaction of short pieces of ribonucleic acid (RNA) with the genetic data -transferring mRNA involving several enzyme complexes. As a result, the mRNA is cleaved into multiple fragments, and the information to be transmitted is destroyed or prevent translation into a protein.
In the life sciences to RNA interference has established itself as an experimental option for the decommissioning of genes ( " gene knockdown "). New based on RNA interference therapies are in clinical development. For the discovery of the mechanism of RNA interference, the two U.S. scientists Andrew Z. Fire and Craig C. Mello received the 2006 Nobel Prize in Physiology or Medicine.
- 3.1 Education interfering RNA
- 3.2 formation of the RNA-induced silencing complex
- 3.3 Activation and function of the RNA-induced silencing complex 3.3.1 Cleavage of the target RNA
- 3.3.2 Inhibition of the translation
- 3.3.3 Inhibition of transcription
- 3.3.4 RNA activation
- 4.1 Basic Research
- 4.2 therapy
Occurrence
The phenomenon of RNA interference can be observed in all kingdoms of eukaryotic organisms including fungi, plants and animals. Therefore, it is assumed that RNA interference is an evolutionarily very old mechanism. Particularly the mechanisms of RNA interference in the biological model organisms Arabidopsis thaliana ( thale cress ), Caenorhabditis elegans and Drosophila melanogaster have been examined. In particular, the nematode C. elegans as a model organism for the study of RNA interference, as can be introduced in these relatively simply constructed organism particularly light interfering RNA on serving as food genetically modified E. coli bacteria and watch with him robust interference effects are.
Some components of the RNA interference machinery, such as the essential function for the RNA - interference Argonauts proteins also occur in prokaryotes. An RNA interference of eukaryotes largely ähnelnder process is the CRISPR mechanism of prokaryotes to defend against intrusion foreign genetic material by viruses.
Function
In nature, different types of interfering RNA occur. Although these different types of interfering RNA share common or related mechanisms, its function part is very diverse.
Virus Defence
In the defense against foreign RNA RNA interference plays in particular in plants play an important role. It thus represents a key component of the plant defense system against RNA viruses dar. At this defense function is the siRNA ( small interfering RNA), which in plant cells as a result of infection with an RNA virus in the duplication ( replication ) of the virus RNA is formed and at the same time the cell is used for detection and destruction of foreign RNA, involved. Many viruses attempt in turn to escape this defense mechanism via inhibition of proteins involved in RNA interference.
Similar mechanisms of RNA interference to fight infection, could also be found in fungi, nematodes and insects. In mammals, the presence of an endogenous siRNA -based defense mechanism is not secured. This task can be performed in mammals of special of cellular miRNA (micro RNA) with a direct inhibitory effect on the replication of viruses.
Regulation of gene expression
The RNA interference plays involving the cell's own miRNA in many multicellular creatures in the regulation of gene expression play an important role. Present in the human organism to about 1000 different miRNAs control the estimated activity of about 30% of the human genome. Of these, among other numerous functions of the immune system are affected. Thus, the regulation of gene activity is given by a similar miRNA important as the regulation by transcription factors.
Also, a cell's own variant of the siRNA, the so-called esiRNA ( endogenous siRNA) is involved in the regulation of gene expression. This occurring in both plants, fungi and animals interfering RNA is not a viral disease, in contrast to exogenous siRNA.
Control of transposons
Another object of that control so-called jumping genes ( transposons ), is particularly noticed by another type of interfering RNA molecules, the piRNA ( PIWI interacting RNA). This function of the RNA interference plays an important role in spermatogenesis, and the development of the embryo in humans.
Mechanism
There are several known each other related mechanisms of RNA interference. In these mechanisms are involved as components of small specialized usually double-stranded RNA molecules, as an RNA-induced silencing complex (RISC), designated enzyme complex and the target RNA. RNA interference may be generally divided into three phases. In the first step in the cell more double stranded RNA molecules with ribonuclease enzymes, such as Dicer and Drosha, cut into short double-stranded RNA fragments. In the second step, these fragments are split into single strands, and a strand of the so-called leading strand '(not to be confused with the leading strand of DNA replication ), is incorporated into the RISC enzyme complex. Ultimately, the leading strand recorded activate the enzyme complex to cleave mRNA complementary in its base sequence to the sequence of Leitstrangs. In this way, the leading strand determines the mRNA is cleaved in what way. Alternatively, the enzyme complex can block the function of a leading strand complementary to the mRNA as an information carrier without cleavage. Both routes can lead to suppression of the conversion of genetic information of a protein in a characteristic.
Education interfering RNA
A first key step of RNA interference is the formation of double-stranded RNA molecules ( dsRNA) having a length of about 20 to 30 base pairs. Depending on their origin can be drawn between different types of short double-stranded RNA molecules.
SiRNA, an approximately 19 to 23 base pair double-stranded RNA with small 3'- terminal two nucleotides overhanging each is formed by a cleavage of a large double-stranded RNA molecule. This precursor RNA can be several hundreds to thousands of base pairs long, and is, for example, at the time of duplication of viral RNA. dsRNA can be not only exogenous but also endogenous origin. At the division in particular is the enzyme Dicer, an RNase III called, involved. This mechanism is especially used for defense against RNA viruses.
Against which the miRNA is encoded by the genomic DNA. In a multistage process, the miRNA is formed from non- protein-coding regions of the DNA. Primary is pri-miRNA (primary miRNA) generated with the help of RNA polymerase II transcription by a single strand of RNA that folds due to considerable palindromic regions to a characteristic secondary structure. In animal cells, the pri-miRNA is cleaved even in the nucleus with the help of an enzyme complex, the Microprocessor complex with the participation of RNase Drosha into hairpin-shaped pre-miRNA ( precursor miRNA ) with a length of 65 to 70 nucleotides. The formed pre-miRNA is finally cut in the cytosol by means of Dicer in a comprehensive each about 21 to 25 base pairs miRNA duplex. In plant cells, however, the formation of a miRNA duplex is done directly from the pri-miRNA with the participation of the localized in the nucleus RNAse DCL1.
A occurring in animal gametes variant of RNA interference using single-stranded piRNA ( PIWI -interacting RNA). The formation of about 24 to 31 nucleotides comprising the interfering RNA molecules differs significantly from that of siRNA, and miRNA. At the not yet fully clarified piRNA biogenesis of either double-stranded RNA precursor molecules still RNases are involved in type III. It is believed among other things that the piRNA is formed after the formation of a primary transcript by transcription of a piRNA cluster in a cycle process known as ping - pong mechanism involving PIWI proteins.
In addition to these interfering RNAs are further capable of RNA interference RNA types, such as RNA and 21U esiRNA known.
Formation of the RNA-induced silencing complex
The central element of RNA interference is referred to as RNA-induced silencing complex (RISC) enzyme complex. The composition of the complex varies depending on the used interfering RNA and RNA Interferenzweg and also may vary from species to species. For the basic functions of the RNA-induced silencing complex proteins of the Argonaute family are responsible. The formation of the RNA-induced silencing complex itself is a multi-step process and takes place in the so-called P- Bodys place in the cytosol.
The first step is formed by Dicer comprehensive 19 to 23 base pairs in double-stranded siRNA or miRNA molecules are transferred to the Argonauts proteins of the RNA - induced silencing complex. This double-stranded RNA -loaded complex is also referred to as a pre -RISC. It is assumed that the interfering RNA enzymes responsible for the formation interact with the RNA-induced silencing complex and mediate the transfer. The interfering RNA can in turn control indirectly, on which Argonauts proteins it is passed. It could be shown by the example of the fruit fly Drosophila melanogaster, that is at least passed in this double-stranded and usually not perfectly paired miRNA by Dicer DCR1 with the double-stranded RNA - binding domain Loquacious ( LOQS ) to the Argonauts protein AGO1. Double- perfectly matched siRNA is transferred in Drosophila melanogaster, however, of Dicer DCR2 with the double-stranded RNA - binding domain of R2D2 to the Argonauts protein AGO2. Thanks to the direct transfer of interfering RNA can also be ensured that no single degradation products of the mRNA get into the RNA-induced silencing complex and thus cause a dysregulation of RNA interference.
In a second step, the double strands of the siRNA or miRNA can be unwound bound and cleaved within the pre- RISC previously formed. The called leading strand single-stranded RNA remains in the RNA-induced silencing complex, which is called in this state holo- RISC and the other strand is degraded and leaves the complex. The selection of highly specific Leitstrangs done it. In RNA - induced silencing complex remains double- strands of the two possible interfering RNA strand that has a lower thermodynamic stability at the 5 ' end.
In the third step, a leading strand complementary to the mRNA is eventually incorporated into the RNA-induced silencing complex. For the binding of the target mRNA is a thermodynamic stability of the leading strand mRNA complex at the 5'- end of the Leitstrangs of particular importance. The inclusion of the mRNA in the complex may also be affected by the mRNA bound to ribonucleoproteins. These can block the binding sites of mRNA or otherwise release due to secondary structures inaccessible binding sites.
Activation and function of the RNA-induced silencing complex
Cleavage of the target RNA
The best-researched consequence of the activation of the RNA-induced silencing complex is the cleavage of a bound in the complex and leading strand complementary to the mRNA. This mechanism, which can be particularly observed in siRNA, presupposes an Argonauts protein with endonuclease activity and a perfect possible complementarity between leading strand and target mRNA. Of the four human Argonaut proteins that are involved in the siRNA or miRNA -mediated RNA interference only has a AGO2 endonuclease. Cleavage of the RNA target can furthermore be observed as a result of piRNA -mediated RNA interference. This splitting is due to the endonuclease activity of participating in the ping-pong cycle PIWI proteins. The cleaved target mRNA can be degraded in the P- Bodys on.
Inhibition of translation
The main function of the RNA - induced silencing complex (especially for the miRNA -mediated RNA interference ) is the inhibition of translation of information of a mRNA into a protein on the ribosomes (translation). For this function, neither endonuclease still a high degree of complementarity between the leading strand and the target mRNA condition. Only nucleotides 2 to 7 of which Leitstrangs must be complementary to the target mRNA. The underlying molecular mechanisms of inhibition of translation are less well researched. At least two possible mechanisms which have already been observed in the cells of Drosophila melanogaster are responsible for the inhibition of the conversion of the mRNA information into a protein. First, the RNA-induced silencing complex can block via protein -protein interactions with Translationsinitialisierungsfaktoren. On the other hand, a reduction of the polyadenylation signal of the mRNA are observed by the activated RNA-induced silencing complex. Both mechanisms have an inhibition of translational initiation.
Inhibition of transcription
In addition to the RNA interference small double-stranded RNA molecules can lead to inhibition of transcription. At this taking place in the cell nucleus called transcriptional gene silencing as a RNA-induced transcriptional silencing complex ( RITS ) designated variant of the RNA-induced silencing complex is involved. The activated RNA-induced transcriptional silencing complex via histone modifications in the formation of heterochromatin regions of the genome. These areas are not accessible to the enzymes of the transcription.
RNA activation
Another traceable to small RNA molecules in addition to the phenomenon of RNA interference is the so-called RNA activation ( RNAA ). As the underlying mechanisms of activation of transcription by the promoter - specific interfering RNA, an activation of the translation involving Argonaut proteins, an interaction with the cell 's own, and miRNA antisense mechanisms are discussed.
Application
Basic research
RNA interference is used in basic research to elucidate the still unknown function of a gene known to be examined and its encoded protein. The RNA interference allows the targeted elimination of any gene. This elimination of the function of the gene encoded by the protein may be derived. It must only the nucleotide sequence of the gene to be examined must be known in order to develop potentially interfering RNA molecules. Thus also called " gene knock -down " referred to application of the RNA interference for studying the function of a gene and its encoded protein is substantially less expensive than the conventional gene knock-out. In addition, the application of RNA interference is time-consuming and more promising than the development of a ligand as an inhibitor of protein function. This is particularly the case when a pharmacological discrimination of closely related proteins, is practically impossible.
Also for the inverted question, the search for those responsible for a known function or a particular characteristic genes or proteins, suitable experimental use of RNA interference. For this purpose, RNA interference libraries are used with interfering RNAs against every single gene that can be used with the aid of high-throughput screening. These so-called genome-wide RNA interference screening can also be found in pharmaceutical research in search of new target molecules for new drugs ( Target Discovery ) application.
For both areas of application of RNA interference in fundamental research in particular synthetic siRNA or shRNA molecules ( short hairpin RNA) are used. They are either introduced directly using various transfection into cells or formed indirectly in the cells after introduction of a vector, such as an shRNA -encoding plasmid or a virus, taking advantage of the cellular transcription. To avoid misinterpretation by the non-specific effects which occur repeatedly in practice, appropriate control experiments are conducted or the specificity is confirmed by several interfering RNAs against one and the same gene.
Therapy
Although the RNA interference is a recently discovered biological mechanism, the first RNA - interference - based therapeutics are already in the late stages of clinical development. This rapid development can be attributed in clinical development on the one hand the potential of RNA interference as a therapeutic method and the other hand on the years of experience with antisense oligonucleotides and ribozymes. The most advanced was the development of Bevasiranib, one directed against the vascular endothelial growth factor ( VEGF) siRNA, which should be used for the treatment of age-related macular degeneration, but failed in a clinical phase III trial. Presented Shortly after this setback, several large pharmaceutical companies, including Hoffmann -La Roche, its siRNA -based development programs a. Despite this, there are other RNA - interference -based therapeutics, as against the VEGF receptor -1 directed Sirna -027 and directed against the gene RTP801 PF -655 for the treatment of age-related macular degeneration, as well as anti- viral nucleocapsid ALN- RSV01 for the treatment of respiratory syncytial virus infections, at least currently in Phase II clinical trials.
Also intrinsic to the cell interfering RNAs represent potential targets for pharmaceutical development dar. So-called Antagomirs that block cell's miRNAs via antisense mechanisms, are in preclinical development.
History of discovery
Around the year 1990, the research team tried to Joseph Mol and Richard Jorgensen, to enhance the flower color of petunias. They intended to bring extra copies of the gene into the plant dihydroflavonol and hoped this would enable the production of flower colors to stimulate ( from the group of flavonoids). However, the opposite was the case. To everyone's surprise, most of the genetically modified plants were less colored than untreated plants, some of them snow-white. First, the term " co-suppression " was coined for this phenomenon, since not only introduced into the plant genes, but also the corresponding naturally occurring in the plant gene dihydroflavonol yielded no or only little functional protein.
Further work showed some years later that the genes were switched off not only at the level of transcription, but that the addition of them produced mRNA was rapidly degraded in the cells - a process of post- transcriptional gene silencing ( PTGS) was baptized. Around the same time, similar phenomena from fungi ( Neurospora crassa ) under the name Quelling from algae and from the nematode C. elegans have been described.
In 1998, it was clear after a series of investigations that the mRNA itself is heavily involved in the phenomenon of PTGS. Almost simultaneously described with the complete sequencing of the genome of C. elegans Andrew Fire and Craig Mello in 1998, the technique of RNA interference ( RNAi), in which double-stranded RNA in C. elegans leads to an efficient and specific gene knockdown. To this end them the Nobel Prize in Physiology or Medicine 2006 was awarded. Exactly how the introduction of double-stranded RNA but results in the organism to degrade the target RNA, was only seen when in 1999 Andrew J. Hamilton and David C. Baulcombe were able to isolate short RNA molecules with a length of about 25 nucleotides in directly related to the regulated RNA is: the siRNA, which gives the RNAi specificity by binding the target RNA through base pairing.
In the attempt to transfer the strategy used by Craig Mello and Andrew Fire on vertebrates, but had subsequently been significant problems: The cells used did not seem to tolerate the long double-stranded RNAs, and it came to programmed cell death (apoptosis). First published in 2001, Sidon Elbashir and Thomas Tuschl a way how this problem can be circumvented. They used short double-stranded RNA with 21 nucleotides that do not lead to apoptosis, but functionally sufficient for gene silencing.
It has been postulated that interfering RNA in the defense against RNA viruses and in the regulation of several mobile genetic elements ( transposons ) could play a role. Therefore, they tried - motivated by the fact that RNA interference is a universal process in eukaryotes probably - to isolate interfering RNA from untreated cells. So they came across another group of small RNA molecules that miRNAs. Two of these miRNAs, lin -4 and let-7 have been previously discovered in C. elegans and initially been referred to as stRNAs (small temporal RNAs).