RNA Interference (RNAi) - Definition, Mechanism, Application

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.

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.

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.

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
https://www.thermofisher.com/blog/ask-a-scientist/what-is-rnai/

https://www.news-medical.net/life-sciences/What-is-RNA-Interference.aspx
https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/
https://geneticeducation.co.in/rna-interference-rnai-a-process-of-gene-silencing/