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In 1989, Sidney Altman and Thomas Cech received a Nobel Prize in Chemistry for the discovery of RNA as a biocatalyst, in addition to being the molecule of heredity. While announcing the award, the Royal Swedish Academy of Sciences made a perceptive comment: "future use of gene shears will require that we learn more about the molecular mechanisms of RNA". The discovery of an evolutionarily conserved mechanism of RNA interference accentuates this objective. RNA interference (RNAi) is an effective post-transcriptional mechanism involving direct mRNA degradation or inhibition of protein translation via double-stranded RNA in a sequence specific manner. Present in all eukaryotes, from yeast to mammals, this system helps determine the active genes and their functioning within the living cells. This method of gene silencing is essentially adapted for both gene expression and regulation of cell growth, and provides considerable defence to host and its genome against parasitic viruses and transposons.
The discovery of RNAi was fairly accidental, when the American scientists Andrew Fire and Craig Mello were performing experiments on the anti-sense RNA. They were awarded the Nobel Prize for Physiology or Medicine soon after their work was published in 1998. Likewise, the genes responsible for RNAi mechanism were discovered in 2000 by scientists Brenda Bass and her colleagues while working on genetically altered strains of roundworm C. elegans. Furthermore, it was determined that 3 of the 7 genes involved in RNAi process belong to smg gene family, which led the molecular biologists to utilize this mechanism as an experimental tool for degradation of mRNA in the living cells of C. elegans and Drosophila. Ever since, there have been numerous experiments on plants and mammalian cell cultures involving the implementation of exogenous ds-RNAs. These being complimentary to the targeted mRNAs, cause the attenuation or suppression of gene expression within the cells. Consequently, these experiments serve as the basis for the study of reverse genetics, research and are enormously beneficial if utilized in drug target validation. With the current knowledge on the genetic etiology of many diseases, RNAi has been targeted as a potential therapeutic tool, both in vivo and in vitro. Several human diseases in animal models have been successfully treated using the RNAi technique. Moreover, a recent report by Grimm et al not only declares an affirmative use of the RNAi technique but also serves as a somber warning that the effectiveness of the RNAi techniques depends on the absolute understanding of the molecular mechanisms involved.
Mechanism of RNA interference:
MicroRNAs and the small interfering RNAs (siRNAs) are most vital in the process of RNA interference. The RNA silencing pathway comprises of two main phases; the first phase involves the recognition and cleavage of long double-stranded RNA (dsRNA) molecules by RNase-III enzyme called Dicer in the cytoplasm. The dsRNAs utilized for this process could either be adapted from an endogenous source such as pre-microRNAs from RNA-encoding genes or a synthetic exogenous source such as infection from foreign viruses. However, when micro-RNAs are utilized, they are first subjected to post-transcriptional modification with the help of a long RNA coding gene called pri-miRNA. This serves as a primary transcript to the miRNA and is processed in the nucleus to form a stem-loop structure of 70 nucleotides called the pre-miRNA. The pre-miRNA comprises of RNase III enzyme called Drosha and dsRNA-binding protein called Pasha. When acted upon by the enzyme Dicer, the dsRNA portion of the pre-miRNA is cleaved. The resultant short interfering RNAs (siRNAs) formed are made up of 21-28 nucleotide duplexes with symmetric 2-3 nucleotide 3' overhangs and forms a part of a 'silencing complex'.
In the second phase, one of the two strands of each siRNA molecule; the guide strand, is assembled into a multiprotein RNA-induced silencing complex (RISC) with the help of an Adenosine triphosphate (ATP) independent activity of the protein components of RISC. After the activation of the RISC complex, the siRNAs direct them towards the homologous target mRNAs via their unwound antisense strand, known as the anti-guide strand. This consecutively triggers the endonucleolytic cleavage of the mRNA by a Slicer enzyme called Argonaute-2 which is a catalytic component of RISC. This protein is localised in specific regions in the cytoplasm called the P-bodies which have acutely high rates of mRNA decay. Additionally, translation of the target mRNA strand is central to the RNAi process, since it can be more effective against non translated targets.
The cleavage of the target mRNA begins at a single site in the middle of the duplex region of the guide siRNA and the target mRNA, about 10 nucleotides upstream of the 5' end of the siRNA. This results in target mRNA degradation and subsequently causes gene silencing. Since the anti-guide strand of the siRNA is conserved within the RISC complex, it is further utilized as a catalyst for the degradation of additional copies of mRNA. Furthermore, RNAi does not necessarily require a complete sequence homology for the target mRNA, with as few as seven consecutive complementary base pairs being adequate enough to bypass RNAi-mediated silencing. As a result, this process is highly effective and advantageous in mammalian cells.
Various components of the RNA silencing pathway are also utilized for the modification of histones, thereby producing abundant heterochromatin in a process referred to as RNA-induced transcriptional silencing (RITS). Carried out by the RITS complex proteins, this process not only aids in downregulating genes pre-transcriptionally, but also forms the basis of genome organisation and structure.
Given that the heterochromatin formation by the RITS complex is not fully understood, the study for the maintainence of existing heterochromatin regions is vital. The RITS proteins form a complex with the siRNAs that are complementary to the native genes and remain stably bound to their methylated histones. These proteins function for the degradation of nascent pre-mRNAs which have been initiated by RNA polymerase, simultaneously with transcription. The formation of the heterochromatin region is Dicer-dependent, since it causes the formation of siRNAs that attack the target transcripts. However, the maintainence of heterochromatin has been indicated to function as a self-reinforcing feedback loop with the incorporation of the new siRNAs formed from the infrequent nascent transcripts by RNA-dependent RNA polymerase (RdRP) into the RITS complexes.
Applications of RNAi in biomedical research for drug development:
A straightforward and practical application of RNAi in research is the knock down of the expression of the target genes and monitoring its consequences. The use of RNAi can accelerate the evaluation of these target genes for the drug development, since it allows the rapid and effective suppression of any protein expression within nearly all cell types. RNAi can also be potentially useful if applied to screen large sets of gene families and target their cell kinases, ion channels or G protein-coupled receptors, in order to locate the starting point for the development of a new drug.
Another new RNAi based research concept for the development of cancer drugs is the genotype-specific drug target. It primarily targets those proteins in the body, whose inactivation causes toxicity only to those cells that carry a cancer-specific genetic lesion. Hypothetically, these drugs would target for the cancerous cells more effectively than the ordinary cytotoxic drugs, since their prevailing specificity towards cancer lesions. RNAi technique can also be particularly used to expose the synthetic lethal interactions; a combination of two non-lethal measures resulting in cell death, within the mammalian cells, with its first screenings recently described in the research sector. However, due to the varied nature of tumours, it has proved to be a rather complicated approach for clinical trials.
The resistance of cells towards therapy often hampers the treatment of diseases, and the cellular mechanisms towards resistance are typically ambiguous. In this case, RNAi is capable of identifying genes involved in drug resistance by simply knocking down genes from drug-sensitive cells in-vitro, exposing them to the appropriate drugs and then evaluating their survival rate. This uncovers the drug sensitive genes, and with the help of clinical trials, the pathways and mechanisms of drug sensitive genes can be authenticated.
Consequently, the siRNA oligonucleotides can be of versatile aid to target genes for suppression as compared to the expensive and prolonged procedure of small-molecule drugs. The main issue that plagues the oligonucleotide based therapy, is how to deliver siRNAs to specific cellular or tissue targets. In the experiments performed by Song and colleagues, they engineered mouse melanoma cells to express GP160 cell surface antigen protein of HIV-1 and implanted these into the host mice. This was followed by the injection of cells consisting of a mixture of siRNA and protamine, a protein that binds DNA, which was designed to target genes regulating the cell cycle (c-myc), apoptosis (mdm2) and angiogenesis (vegf). This mixture inhibited the establishment of tumours expressing the surface antigen GP160, but was ineffective against the remainder.