| RNA Interference - A Regulatory Mechanism in a | | | | have been found to be analogus to the RNAi |
| Living Cell | | | | mechanism in eukaryotes. DNA of many bacteria and |
| Imagine a situation where your cell fails to control the | | | | archaea are found to consist of direct repeats |
| amount of protein being produced or the type of | | | | ranging in size from 24 to 48 base pairs known as |
| protein being produced. This may lead to a deadly | | | | CRISPR. The repeats show some dyad symmetry |
| disease. But nature has equipped your body with | | | | and are separated by spacers of similar length. |
| regulatory mechanisms to check this as and when | | | | Spacer sequences generally have a unique genome |
| required. One such regulatory mechanism is RNA | | | | and some spacer sequences usually match the |
| interference (RNAi), also known as post | | | | sequences in phage genomes. It has been recently |
| transcriptional gene silencing and quelling. | | | | demonstrated that, these spacers protect the cell |
| Andrew Fire and Craig Mello published their | | | | from infection. |
| break-through study on the mechanism of RNA | | | | 3 Importance of RNAi mechanism |
| interference in Nature in 1998 [1]. | | | | 3.1 Defense mechanism in plants |
| 1 Why do you need something like | | | | Plants show an adaptive immune response against |
| RNAi mechanism? | | | | viruses and other foreign genetic material through this |
| DNA and RNA, are biopolymers and the sequence of | | | | mechanism. Plants such as Arabidopsis thaliana, |
| their monomer subunits carries information for the | | | | express multiple dicer homologs which specifically act |
| proper cell functioning. The information, for the | | | | against different viruses. In some cases, plant |
| production of the required proteins is coded in DNA | | | | genomes also express endogenous siRNAs in |
| which gets transcribed to RNA and is ultimately | | | | response to bacterial infection. |
| translated into proteins. To make a living cell function | | | | Among animals, Drosophila, shows antiviral innate |
| properly, a cell needs to control both the type of the | | | | immunity against pathogens such as Drosophila X |
| gene and the quantity of the gene to be activated | | | | virus, through RNAi mechanism. |
| at a particular time. RNA interference (RNAi) is a part | | | | 3.2 Regulation of genes |
| of this control mechanism which is an outcome of | | | | 3.2.1 Downregulation |
| post transcriptional gene silencing and acts at the | | | | Endogenously expressed miRNAs play a significant |
| level of RNA. | | | | role in: |
| The molecules contributing to RNA interference are: | | | | - Translational repression. |
| - microRNA (miRNA) - small RNA molecules | | | | - Regulation of development – more specifically |
| - siRNA – small interfering RNA | | | | timing of morphogenesis. |
| 2 Mechanism of RNA interference in | | | | - Maintenance of incompletely differentiated cell types |
| a cell | | | | such as stem cells |
| There are basically two dsRNA (double stranded | | | | In plants, mainly genes of transcription factors are |
| RNA) pathways, exogenous and endogenous, which | | | | regulated by miRNAs. |
| finally converge at the RISC complex. | | | | 3.2.2 Upregulation |
| 2.1 Exogenous pathway | | | | RNA sequences (siRNA and miRNA) that are |
| During an exogenous pathway, dsRNA (coming | | | | complementary to parts of a promoter are dubbed |
| from infection by a virus with an RNA genome or | | | | which in turn increase gene transcription. |
| laboratory manipulations), gets directly imported into | | | | 3.2.3 Maintenance of genome stability |
| the cytoplasm. The imported dsRNA, activates a | | | | In the case of C. elegans and plants, RNAi mechanism |
| member of the RNase III family of dsRNA-specific | | | | blocks the action of transposons (mobile elements in |
| ribonucleases protein, Dicer, within the cytoplasm. The | | | | the genome) and maintains the genome stability. |
| Dicer further cleaves dsRNAs, to small 20-25 | | | | 3.3 Technological applications |
| base-paired double-stranded fragments with a few | | | | 3.3.1 Facilitating Gene-knockdown |
| unpaired 2-nucleotide 3' overhangs on each end [2]. | | | | To study the physiological effect, of a target gene in |
| These Dicer-induced small double-stranded fragments | | | | vivo a double stranded RNA, complementary to the |
| are called “small interfering RNAs” (siRNAs). | | | | target gene is introduced into the cell or organism. |
| Further, siRNAs get separated into single strands | | | | This is recognized as exogenous genetic material and |
| followed by integration into an active RNA-induced | | | | activates the RNAi pathway, resulting into drastic |
| silencing complex (RISC). The siRNAs integrated into | | | | decrease in the expression of a targeted gene. This |
| the RISC complex, base-pair to their target mRNA | | | | technique is different from knock out technique, |
| and induce cleavage of the mRNA. This prevents | | | | wherein the expression of gene is entirely eliminated. |
| the target mRNA from being translated. | | | | 3.3.2 Application in functional genomics |
| | | | Many plant genomes, have more than two |
| 2.2 Endogenous pathway | | | | homologous sets of chromosomes (polyploid) and |
| During an endogenous pathway of RNA interference, | | | | tracing the location of a particular gene and its related |
| in which pre-miRNAs play an active role, dsRNA | | | | function is challenging with the traditional genetic |
| originates within the cell. Primary transcripts known | | | | engineering methods. This problem is solved by the |
| as pre-microRNA (pre-miRNA) are produced by a set | | | | RNAi mechanism. |
| of RNA coding genes in the genome. These | | | | 3.3.3 Medical application |
| pre-miRNAs get processed to 70-nucleotide stem | | | | The introduction of siRNAs, has been found to be |
| loop structures by the microprocessor complex, | | | | very useful in the treatment of diseases like macular |
| within the nucleus, further getting exported to the | | | | degeneration and respiratory syncytial virus in case of |
| cytoplasm to be cleaved by Dicer. The pre-miRNAs | | | | mammals. RNAi mechanism is also used as an |
| undergo extensive post-transcriptional modification, to | | | | antiviral therapy against diseases caused by herpes |
| generate mature miRNAs, structurally similar to | | | | simplex virus type 2, hepatitis A, hepatitis B. |
| siRNAs produced from exogenous dsRNA. | | | | RNAi-mechanism governs gene regulation in |
| 2.3 What differentiates the working | | | | transgenic organisms, suggesting its role in gene |
| mechanism of siRNAs from miRNAs? | | | | therapy. |
| The difference in the working mechanism of siRNAs | | | | 3.3.4 Biotechnological application |
| and miRNAs lies in their specificity. The miRNAs, | | | | To reduce the levels of natural toxins in food plants |
| especially those in animals, show a lesser specific RNA | | | | you can use a stable, heritable and specific siRNA |
| interference. They show an incomplete base pairing | | | | against the toxin. For example: |
| to a target and inhibit the translation of many | | | | - Cotton seeds are rich in dietary proteins but |
| different mRNAs with similar sequences. In contrast, | | | | unpalatable by humans as they contain a natural toxic |
| siRNAs are very specific in base-pairing and induce | | | | terpenoid product, called gossypol. RNAi mechanism |
| mRNA cleavage only at a single and specific target. | | | | has been used to reduce the levels of delta-cadinene |
| 2.4 Role of RISC complex | | | | synthase, an enzyme essential for the production of |
| The RNA-induced silencing complex (RISC) is made | | | | gossypol. |
| up of endonucleases called argonaute proteins. These | | | | - Cassava plants produce cyanogenic natural product, |
| proteins, are localized to specific regions in the | | | | linamarin, and RNAi mechanism has been used to |
| cytoplasm called P-bodies (or cytoplasmic bodies or | | | | reduce its levels. |
| GW bodies), which are regions with high rates of | | | | 4 Conclusion |
| mRNA decay. A separation of the two strands of | | | | RNAi machinery is like a weapon for the cells and |
| siRNA is performed by the protein components of | | | | helps them in defending against parasitic genes like |
| RISC complex. One of the two strands of siRNA | | | | viruses and transposons. It regulates development of |
| known as the “guide strand”, binds the | | | | an organism and proper function of its cells and |
| argonaute protein, thereby facilitating these proteins | | | | tissues, as well as gene expression within the |
| to cleave the target mRNA strand complementary to | | | | organism. RNAi is the latest experimental approach, |
| the bound siRNA. The other strand of siRNA known | | | | used to detect the function and location of the gene. |
| as anti-guide strand or passenger strand is degraded | | | | It also leads us to new applications in medicine. |
| during RISC activation. | | | | 5 References |
| 2.5 Interference mechanism in eukaryotes | | | | [1] Fire A, Mello CC. Potent and specific genetic |
| and prokaryotes | | | | interference by double-stranded RNA in |
| The RNAi mechanism is found in many eukaryotes | | | | Caenorhabditis elegans. Nature. 1998 Feb 19;391 |
| including animals. The regulatory RNAs, in case of | | | | (6669):806-11. |
| prokaryotes are not analogous to miRNAs, as the | | | | [2] Vermeulen A, Reynolds A. The contributions of |
| dicer enzyme is not involved. CRISPR (Clustered | | | | dsRNA structure to Dicer specificity and efficiency. |
| Regularly Interspaced Short Palindromic Repeats) | | | | RNA. 2005 May;11(5):674-82. |
| systems, providing acquired immunity in prokaryotes, | | | | |