Posts Tagged "Interference"
RNA Interference
RNA interference (RNAi) or double-stranded RNA (dsRNA) is a system within living cells that helps to control which genes are active and how active they are. siRNAs were first discovered by David Baulcombe’s group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants1 and later independently identified in wide variety of eukaryotic organisms. These dsRNAs are rapidly processed into short RNA duplexes of 21 to 28 nucleotides in length, which then guide the recognition and ultimately the cleavage of complementary single-stranded RNAs, such as messenger RNAs or viral genomic/antigenomic RNA (Fig. 1). According to their origin or function, naturally occurring small RNA have been described: short interfering RNAs (siRNA), repeat-associated short interfering RNA (rasiRNA or shRNA) and microRNA (miRNA). RNA interference has many biological functions – it is a vital part of the immune response against viruses and also downregulates gene expression by transcriptional silencing of genes or upregulates promoting by RNA activation. Finally, artificial introduction of long dsRNA or siRNA has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms.http://www.biosyn.com/TEWdetail.aspx?TEWid=180
A biochemical understanding of the RNAi pathway was crucial to realizing that dsRNAs shorter than 30 base pairs (bp) could be used to trigger an RNAi response in mammals. Tuschl and colleagues showed that transfection of mammalian cells with short RNAs could induce the sequence-specific RNAi pathway, and so overcame the barrier to the use of RNAi as a genetic tool in mammals2. The impetus to use siRNAs and other small RNAs in mammalian cells also came from the long-standing view that protein kinase receptor (PKR) activation3 and similar responses were not effectively triggered by short dsRNAs. Following the initial reports, it took a remarkably short period of time for siRNAs triggers to be adopted as a standard component of the molecular biology toolkit. siRNAs can be introduced into mammalian cells using a variety of standard transfection methods. The strength and duration of the silencing response is determined by several factors: on a population basis, the silencing response is affected mainly by the overall efficiency of transfection, which can be addressed by optimizing conditions. In each cell, silencing depends on the amount of siRNA that is delivered and on the potential of each siRNA to suppress its target, or its potency. Even a relatively impotent siRNA can silence its target provided that sufficient quantities of the siRNA are delivered. However, essentially ‘forcing’ the system by delivering large amounts of reagent is likely to lead to numerous undesired effects.
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Rna Interference: Imgenex Launched the Psuppressoradeno Construction Kit for Adenovirus Mediated Gene Knockdown
RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has been observed in all eukaryotes, from yeast to mammals. The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements that use double-stranded RNA (dsRNA) in their life cycles. They have also been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in regulation of translation, transcription, chromatin structure, and genome integrity. In plants and animals, RNA silencing has been adapted to play a critical role in regulation of cell growth and differentiation using a class of small RNAs. In the RNA interference process, the dsRNAs get processed into 20-25 nucleotide (nt) small RNAs by an RNase III-like enzyme called Dicer. Then, the small RNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The small RNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The small RNAs that provide target specificity to the silencing machinery includes short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and microRNAs (miRNAs) and is distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly base-paired RNA, while miRNAs originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now been recognized. The endogenous siRNAs are derived from repetitive sequences within the genome, and are termed repeat-associated siRNAs, or rasiRNAs. miRNAs were discovered through their critical roles in development and cellular regulation, and represent a large class of evolutionarily conserved RNAs. miRNAs have always been recognized as being of endogenous origin. RNA interference has emerged as a natural mechanism for silencing gene expression over the past decade. This ancient cellular antiviral response can be harnessed to allow specific inhibition of the function of any chosen target genes, including those involved in causing diseases such as cancer, AIDS, and hepatitis. It is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Last but not the least the technology can be harnessed as a novel therapeutic agent and is suitable for combating viral diseases, cancers and inflammatory diseases.
Imgenex (San Diego) recently launched the pSuppressorAdeno construction kit for adenovirus mediated gene knockdown. The kit provides the ability to infect a broad range of cell types, including many primary cell lines as well as dividing and nondividing cells, according to a company official. The kit also offers the flexibility to validate sequences using the nonviral expression plasmid prior to construction of adenoviruses, notes Sujay K. Singh, Ph.D., president and CEO of Imgenex, which markets plasmid-based RNA interference (RNAi) products. “One of the greatest advantages is the ability of recombinant adenovirus vectors to reduce gene expression both in vitro and in vivo,” he adds. RNAi, initially considered a bizarre attribute of petunias and later a gene-silencing mechanism in worms, is creating a stir as one of the hottest new technologies in molecular biology. It is revolutionizing the field of functional genomics.
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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.
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