Posts Tagged "Dicer"
RNA Interference – A Regulatory Mechanism in a Living Cell
RNA Interference – A Regulatory Mechanism in a Living Cell
Imagine a situation where your cell fails to control the amount of protein being produced or the type of protein being produced. This may lead to a deadly disease. But nature has equipped your body with regulatory mechanisms to check this as and when required. One such regulatory mechanism is RNA interference (RNAi), also known as post transcriptional gene silencing and quelling.
Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA interference in Nature in 1998 [1].
1 Why do you need something like RNAi mechanism?
DNA and RNA, are biopolymers and the sequence of their monomer subunits carries information for the proper cell functioning. The information, for the production of the required proteins is coded in DNA which gets transcribed to RNA and is ultimately translated into proteins. To make a living cell function properly, a cell needs to control both the type of the gene and the quantity of the gene to be activated at a particular time. RNA interference (RNAi) is a part of this control mechanism which is an outcome of post transcriptional gene silencing and acts at the level of RNA.
The molecules contributing to RNA interference are:
microRNA (miRNA) – small RNA molecules siRNA – small interfering RNA 2 Mechanism of RNA interference in a cell
There are basically two dsRNA (double stranded RNA) pathways, exogenous and endogenous, which finally converge at the RISC complex.
2.1 Exogenous pathway
During an exogenous pathway, dsRNA (coming from infection by a virus with an RNA genome or laboratory manipulations), gets directly imported into the cytoplasm. The imported dsRNA, activates a member of the RNase III family of dsRNA-specific ribonucleases protein, Dicer, within the cytoplasm. The Dicer further cleaves dsRNAs, to small 20-25 base-paired double-stranded fragments with a few unpaired 2-nucleotide 3′ overhangs on each end [2]. These Dicer-induced small double-stranded fragments are called “small interfering RNAs” (siRNAs). Further, siRNAs get separated into single strands followed by integration into an active RNA-induced silencing complex (RISC). The siRNAs integrated into the RISC complex, base-pair to their target mRNA and induce cleavage of the mRNA. This prevents the target mRNA from being translated.
2.2 Endogenous pathway
During an endogenous pathway of RNA interference, in which pre-miRNAs play an active role, dsRNA originates within the cell. Primary transcripts known as pre-microRNA (pre-miRNA) are produced by a set of RNA coding genes in the genome. These pre-miRNAs get processed to 70-nucleotide stem loop structures by the microprocessor complex, within the nucleus, further getting exported to the cytoplasm to be cleaved by Dicer. The pre-miRNAs undergo extensive post-transcriptional modification, to generate mature miRNAs, structurally similar to siRNAs produced from exogenous dsRNA.
2.3 What differentiates the working mechanism of siRNAs from miRNAs?
The difference in the working mechanism of siRNAs and miRNAs lies in their specificity. The miRNAs, especially those in animals, show a lesser specific RNA interference. They show an incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs are very specific in base-pairing and induce mRNA cleavage only at a single and specific target.
2.4 Role of RISC complex
The RNA-induced silencing complex (RISC) is made up of endonucleases called argonaute proteins. These proteins, are localized to specific regions in the cytoplasm called P-bodies (or cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay. A separation of the two strands of siRNA is performed by the protein components of RISC complex. One of the two strands of siRNA known as the “guide strand”, binds the argonaute protein, thereby facilitating these proteins to cleave the target mRNA strand complementary to the bound siRNA. The other strand of siRNA known as anti-guide strand or passenger strand is degraded during RISC activation.
2.5 Interference mechanism in eukaryotes and prokaryotes
The RNAi mechanism is found in many eukaryotes including animals. The regulatory RNAs, in case of prokaryotes are not analogous to miRNAs, as the dicer enzyme is not involved. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, providing acquired immunity in prokaryotes, have been found to be analogus to the RNAi mechanism in eukaryotes. DNA of many bacteria and archaea are found to consist of direct repeats ranging in size from 24 to 48 base pairs known as CRISPR. The repeats show some dyad symmetry and are separated by spacers of similar length. Spacer sequences generally have a unique genome and some spacer sequences usually match the sequences in phage genomes. It has been recently demonstrated that, these spacers protect the cell from infection.
3 Importance of RNAi mechanism 3.1 Defense mechanism in plants
Plants show an adaptive immune response against viruses and other foreign genetic material through this mechanism. Plants such as Arabidopsis thaliana, express multiple dicer homologs which specifically act against different viruses. In some cases, plant genomes also express endogenous siRNAs in response to bacterial infection.
Among animals, Drosophila, shows antiviral innate immunity against pathogens such as Drosophila X virus, through RNAi mechanism.
3.2 Regulation of genes 3.2.1 Downregulation
Endogenously expressed miRNAs play a significant role in:
Translational repression. Regulation of development – more specifically timing of morphogenesis. Maintenance of incompletely differentiated cell types such as stem cells
In plants, mainly genes of transcription factors are regulated by miRNAs.
3.2.2 Upregulation
RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter are dubbed which in turn increase gene transcription.
3.2.3 Maintenance of genome stability
In the case of C. elegans and plants, RNAi mechanism blocks the action of transposons (mobile elements in the genome) and maintains the genome stability.
3.3 Technological applications 3.3.1 Facilitating Gene-knockdown
To study the physiological effect, of a target gene in vivo a double stranded RNA, complementary to the target gene is introduced into the cell or organism. This is recognized as exogenous genetic material and activates the RNAi pathway, resulting into drastic decrease in the expression of a targeted gene. This technique is different from knock out technique, wherein the expression of gene is entirely eliminated.
3.3.2 Application in functional genomics
Many plant genomes, have more than two homologous sets of chromosomes (polyploid) and tracing the location of a particular gene and its related function is challenging with the traditional genetic engineering methods. This problem is solved by the RNAi mechanism.
3.3.3 Medical application
The introduction of siRNAs, has been found to be very useful in the treatment of diseases like macular degeneration and respiratory syncytial virus in case of mammals. RNAi mechanism is also used as an antiviral therapy against diseases caused by herpes simplex virus type 2, hepatitis A, hepatitis B. RNAi-mechanism governs gene regulation in transgenic organisms, suggesting its role in gene therapy.
3.3.4 Biotechnological application
To reduce the levels of natural toxins in food plants you can use a stable, heritable and specific siRNA against the toxin. For example:
Cotton seeds are rich in dietary proteins but unpalatable by humans as they contain a natural toxic terpenoid product, called gossypol. RNAi mechanism has been used to reduce the levels of delta-cadinene synthase, an enzyme essential for the production of gossypol. Cassava plants produce cyanogenic natural product, linamarin, and RNAi mechanism has been used to reduce its levels. 4 Conclusion
RNAi machinery is like a weapon for the cells and helps them in defending against parasitic genes like viruses and transposons. It regulates development of an organism and proper function of its cells and tissues, as well as gene expression within the organism. RNAi is the latest experimental approach, used to detect the function and location of the gene. It also leads us to new applications in medicine.
5 References
[1] Fire A, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391 (6669):806-11.
[2] Vermeulen A, Reynolds A. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA. 2005 May;11(5):674-82.
Post Graduate in Bio-Chemistry and PG Diploma in Bio-informatics. Worked as a Bio-informatics professional for about 2 years and moved on to a home-based job since.
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How Short Is Short In Rnai Research
The original work of Mello and Fire from the Univ. of Massachusetts demonstrated in C. elegans, that gene expression is controlled by RNA interference (RNAi) . The initial patent applications filed by the Univ. of Massachussetts, with both Mello and Fire as the inventors, and covering this use of RNAi, clained only dsRNA longer than 25 bp’s. Later on, work done in mamalians showed that the long ds RNA was capable of inducing the release of IFN and other pro-inflammatory cytokines, causing dangerous reactions in the animals we tested. We now know that long dsRNA is recognized in the endosomes by TLRs, inducing an undesirable immune response in the animals; a situation that created a new challenge in the study of RNAi. Subsequently other researchers, such as Tuschl et.al. now at Rockefeller Univ, realized that shorter dsRNAi fragments of 25bp of smaller, while still capable of inhibiting the expression of a targeted gene, failed to induce an innate immunity response. In other words, the mammalian system is still capable of utilizing the diced dsRNA produced by the enzyme Dicer, which normally chops down the long dsRNA to sizes of 21-23 nts with 2 bases overhanging at the 3’ends of each strand. These 2 bases overhanging in dsRNA suggests that perhaps Dicer cleaves the long dsRNA in a fashion analogous to restriction enzymes. This short dsRNA can then interact with the RISC complex, where the guide strand is prepared and readied up to base pair with the target mRNA for its cleavage.
The RNAi situation is a good example of the unexpected in science. Although at the time of the initial discovery it was hard to predict that very small fragments of RNA could be pivotal in such important newly found mechanism, currently, even shorter dsRNA fragments, e.g. 15-18 bps’ are being tested. These new third generation modifiers such as LNA’s, UNA’s and others, because of their size have significant therapeutic potential.
There is a significant amount of ongoing research to elucidate the fine details of this novel gene control mechanism, including but not limited to studies of how miRNA precursors are transported to specific compartments of the cell, as these events may play important roles in the processing of the precursor by Dicer to render the active mature form of dsRNA.
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