Table of Contents
RNA Interference Definition
RNA interference is the process by which RNA molecules suppress gene expression by neutralising the targeted messenger RNA molecules.
- RNA interference is an evolutionarily conserved mechanism that is triggered by double-stranded RNA and employs the gene’s own DNA sequence to silence it. This is referred to as gene silencing.
- It is a gene regulatory system that restricts transcript levels in two ways.
- Restricting transcription (transcriptional gene silence) and degrading RNA production (post-transcriptional gene silencing)
- Andrew Z. Fire and Craig C. Mello, two American scientists, found the mechanism in C.elegans cells. They blocked the expression of specific genes by introducing short lengths of double-stranded RNA into the C. elegans cells.
RNA Interference Mechanism
The RNA interference mechanism can be explained in the following steps:
- With the aid of an enzyme called Dicer, long double-stranded RNA is cut into minute bits. These bits are known as small interfering RNA or siRNA.
- Through the RNA-induced silencing complex, the siRNAs pass. The duplex unwinds, activating the RNA. These complexes impede translation and increase RNA breakdown.
- The siRNA binds to the Argonaute protein and removes one of the double-stranded strands. The strand that remains binds to mRNA target sequences. Either the Argonaute protein cleaves the mRNA or recruits other components to control the target sequence.
Why RNAi as a genetic tool?
- Synthetic dsRNA complementary to our target mRNA is designed and injected into the cell line using expression vectors, much like siRNA.
- Once it has been correctly introduced into a cell, the next steps are carried out by the cell’s RNA interference system.
- The dicer identifies the external dsRNA and cleaves it into 21 to 23nt dsRNA fragments.
- It is processed by Dicer and transferred to the cytoplasmic RISC, where the Ago2 protein binds to the siRNA fragments.
- The passenger strand of the siRNA fragments, which is identical to the mRNA, is eliminated, leaving the directed strand in the complex.
- The RISC then migrates the mRNA to its complementary mRNA, attaches to it, and destroys it.
- The inability of mRNA to be translated into protein reduces gene expression.
- The siRNA is a crucial component of in vitro RNAi research; its length is typically 21 nucleotides and is referred to as 21mer.
- The 21mer siRNA is more specific and performs exceptionally well in experiments; however, recent studies imply that the 27mer siRNA is more efficient.
- The 27mer is correctly cleaved by the dicer, resulting in a 2 nucleotide overhang at its 3′ terminus.
- This siRNA resembles the endogenous microRNA more closely. Consult our article on siRNA: Small Interfering RNA (siRNA): Structure And Function
- In in vitro RNA interference investigations, another molecule known as short hairpin RNA (shRNA) is also utilised.
- Chemical modification can improve the efficacy and specificity of the siRNA in the RNA interference (RNAi) process by enhancing the sequence specificity and subsequently reducing the capacity for cross-hybridization.
- Several features must be present in the chosen synthetic nucleic acid (siRNA or shRNA) for RNAi to respond more correctly.
RNA Interference Applications
- The usage of synthetic dsRNA molecules triggers the RNA interference response of a cell and regulates the expression of genes.
- Thus, artificially induced RNA interference has a wide range of applications in the clinical, medicinal, and other research sectors.
- It is now commonly utilised in gene knockout research.
- Additionally, it is utilised in genomics research and investigations. It is currently utilised therapeutically against viral infections, cancer, and neurological illnesses, with researchers intending to use it as a safer treatment for curing ailments.
- RNAi therapeutics can also be utilised in personalised medicine and gene therapy with a specific target.
- In recent years, RNAi technology has become increasingly prevalent in plant research and crop enhancement.
- Scientists are currently utilising RNA interference and antisense RNA in crop development. Using the current technologies, new plant characteristics and disease-resistant plant species are being developed.
- In addition, it is utilised for pest control and crop enhancement. Flvr Savr tomato, decaffeinated coffee, and nicotine-free tobacco are a few of the most notable examples of plant species created via RNAi technology.
- RNAi is also utilised for disease and pathogen resistance, male sterility development, and functional genomic research in plants. Using RNA interference (RNAi), virus-resistant plant species against Banana Bract Mosaic Virus, Rice Tungro Bacilliform Virus, Tobacco Mosaic Virus, and Cucumber Mosaic Virus are produced.
- Artificially designed dsRNA complementary to viral RNA is inserted into the plant genome, mimicking the natural si/miRNA and destroying viral dsRNA whenever it attacks.
- Xu W, Jiang X, Huang L. RNA Interference Technology. Comprehensive Biotechnology. 2019:560–75. doi: 10.1016/B978-0-444-64046-8.00282-2. Epub 2019 Jul 31. PMCID: PMC7152241.
- Kim, D. H., & Rossi, J. J. (2008). RNAi mechanisms and applications. BioTechniques, 44(5), 613–616. doi:10.2144/000112792
- Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA Interference: Biology, Mechanism, and Applications. Microbiology and Molecular Biology Reviews, 67(4), 657–685. doi:10.1128/mmbr.67.4.657-685.2003