Gene silencing by RNA interference

What is RNAi?

RNA interference (RNAi) It is a natural mechanism for controlling gene expression.¹ Also commonly called: Post-transcriptional gene silencing (PTGS).² The RNAi pathway, which is common to plants, animals, fungi, and some bacteria, is thought to be evolutionarily ancient. Defense mechanism against viruses.³ After discovering the RNAi mechanism, molecular biologists applied it in the following ways. Tools for gene silencing.⁴

How does RNAi work?

Natural RNAi mechanisms are directed by either: Short interfering RNA (siRNA) or microRNA (miRNA).¹ There are four key steps in the RNAi pathway.

1. The double-stranded RNA (dsRNA) precursors of siRNA and miRNA bind and cleave them to an endoribonuclease protein called Dicer. Short section of approximately 20 nucleotides.³
2. These small dsRNA fragments bind to the Argonaute protein. Argonaute selects and retains one strand of dsRNA as the guide strand; RNA-induced silencing complex (RISC).⁵
3. The guide strand then locates and binds to complementary target Messenger RNA (mRNA) via Watson-Crick base pairing.¹
4. Once a guide is paired with a target, actions vary depending on the type of guide. For miRNAs, RISC blocks mRNA translation. When guided by siRNA, RISC cleaves target transcripts. Both of these actions subsequently degrade the mRNA.⁶ RNAi is effective and specific by cleaving the target mRNA or preventing its translation into mature protein. silence genes expression.⁷

When miRNAs act as guides for RNAi, only short sections of molecules known as miRNAs act as guides for RNAi. seed areapairing with the target rather than the complete approximately 20 nucleotide sequence.¹ This means that the miRNA guide can target any mRNA with homology to the seed region, even if the remaining sequences do not match. This promiscuous binding activity means that miRNAs can act on many different mRNAs and potentially silence hundreds of genes simultaneously.

In contrast, when RNAi is induced by siRNA, the entire sequence pairs with the target strand with high complementarity. This often results in 100% match between guide and target and very low off-target activity. Additionally, siRNA induces gene silencing of: Directs histone and DNA methylationinactivates chromatin and suppresses transcription.¹

RNAi knockdown

Scientists use RNAi in the lab to Selectively suppress target gene expressiona technique known as gene knockdown.⁸ By delivering dsRNA complementary to the target gene’s mRNA, researchers redirect the RNAi pathway from its standard function to cleave the target and prevent its translation. Unlike complete and permanent gene knockout, RNAi-based gene knockdown is partial and temporary.

Researchers have successfully used RNAi to perform gene knockdown in a variety of organisms and cells, including: Mammalian cell lines, primary cells, stem cells.⁹ Functional genomics researchers now commonly use RNAi to knock down genes and determine their function, including their role in disease.

RNAi technology and its components have won two Nobel Prizes in Physiology and Medicine. The first award was presented in 2006 to Andrew Z. Fire and Craig Mello in 1998. nature In this publication, we use dsRNA Manipulate gene expression in model organisms Caenorhabditis elegans.¹⁰ The second was awarded to Victor Ambros and Gary Ruvkin in 2024 for their discovery of miRNAs and their role in PTGS.

RNAi applications

Researchers use RNAi-based gene knockdown for a variety of applications. In agriculture, scientists are turning to RNAi as a next step. Control of plant pathogensremove viruses, fungi, etc., and ensure more sustainable crop production.¹¹ They are also species-specifically developed; environmentally friendly pesticides RNAi technology can be used, for example, to target essential genes in pests to cause mortality.¹²

Scientists also use RNAi to create disease models and characterize gene function across a variety of medical research areas, including: Infectious diseases, cancer, neurodegenerative diseases.⁸ high throughput Genome-wide RNAi screening This has allowed researchers to identify many novel genes involved in the development of the disease.⁹

Because many genetic diseases are caused by the overexpression of specific genes or the accumulation of toxic mRNA transcripts within cells, medical researchers could also use RNAi to develop new treatments for these diseases. can. For example, RNAi could potentially be used therapeutically. huntington’s diseasewhich is caused by the expansion of a trinucleotide repeat in one allele of . hunting chin gene.¹³ By enabling specific targeting, Mutated mRNA transcript onlyRNAi-based treatments deplete mutated transcripts while leaving intact non-mutated transcripts that are important for normal brain function.¹³

In 2018, the FDA approved the first RNAi-based therapy, a drug known as Patisiran. Designed to treat hereditary transthyretin amyloidosis, patisiran uses siRNA Directs the RNAi pathway to the target transthyretin (TTR) inhibits the production of gene transcripts and mutant proteins.¹⁴