RNAi Mechanism

By Jeyashree Sundaram, MBA

RNA interference (RNAi) is a biological process that inhibits the expression of target genes in the cells of animals, plants, and fungi, and hence, researchers call this process gene silencing.

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RNAi is mostly observed in eukaryotic cells, where double stranded RNA (dsRNA) is used for transcriptional (gene level) and post-transcriptional (RNA level) gene silencing. Gene silencing prevents protein synthesis, and helps to prevent cancers, viral infections, and neurodegenerative diseases.

Functions of RNAi

RNA interference takes place when a long dsRNA is either artificially or naturally introduced in the cell.

The dsRNA is cleaved into small fragments of RNA such as microRNA (miRNA) and simple interfering RNA (siRNA) by Dicer; an endoribonuclease enzyme that is based on the long dsRNA origin and can be both exogenous and endogenous.

Double stranded RNAs can be produced by RNA-dependent RNA polymerase (RdRP), bidirectional transcription involving transposable elements, or artificially in the laboratory.

The process of gene silencing through RNAi takes place in two stages.

Stage one: initiation

The initiation stage involves the generation of siRNA which is then processed by the type III endonuclease Dicer.

There are two types of RNase III (Dicer) enzymes;  Dicer-1, or Dcr-1/Loquacious-PB (Loqs), and Dicer-2, or Dcr-2/R2D2. Although Dcr-1 and Dcr-2 show different characteristics such as substrate specifications and ATP requirements, they are structurally homologous.

miRNA

Dicer-1 does not rely on adenosine triphosphate (ATP) for its function and itsaffinity toward the miRNA precursor. It uses protein partner to bind dsRNA.

In Drosophila, Dcr-1 is found to interact  with the dsRNA binding domain (dsRBD) within a protein called Loqs. Immunoaffinity purification studies have shown  that the Loqs protein present in the precursor miRNA functional processing complex triggers and directs the particular activity that processes miRNA.

These results lead to miRNA-regulated gene expression, which is due to the mediation of miRNA biogenesis by Loqs.

The Loqs gene encodes three dsRBDs that can produce two types of proteins; PA and PB. It has been observed that PB enhances Dcr-1 affinity toward the precursor of miRNA.

siRNA

In contrast, Dicer-2 is ATP-dependent. It also show substrate specificity towards dsRNA.

The structure of Dcr-2 is analogous to Dcr-1, and it also requires a dsRNA binding protein. This protein is R2D2, which functions in combination with particular RNase enzyme and leads to the generation of heterodimeric complex.

Compared with Loq binding protein, R2D2 is only made up of one dsRNA binding domains. The binding domain of R2D2 interacts with the long dsRNA but cannot control the siRNA generation activity of Dicer-2. These results show that both the dsRNA binding domains of R2D2 and Dcr-2 are essential for R2D2/Dcr-2 complex to load and bind siRNA into si-RNA induced silencing complex (siRISC).

Stage two: the effector stage

The second stage of RNAi mechanism involves guide strand (duplex of siRNA and miRNA) incorporation. Guide strands are single strand duplexes of the miRNA and siRNA, and are integrated into effecter complexes that are involved in RNA silencing. These effecter complexes include the RNA-induced initiation of transcriptional gene silencing (RITS) or the RNA-induced silencing complex (RISC).

RISC

RISC is comprised of PPD proteins (the proteins of the PAZ PIWI domain).  PIWI is composed of 300 amino acids and is positioned at the C-terminal, whereas PAZ comprises only 100 amino acids and is centrally located in the protein complex.

Ago2 and hAgo 2 are the  PPD proteins involved in siRNA-mediated silencing in nematodes (C. elegans), arthropoda, and chordata. These proteins are efficient in performing siRNA-mediated m-RNA cleavage.

Research has indicated that a small variation in the siRNA structure affects the RISC complex guided strand determination, and concludes that the enzyme governing the RISC assembly chooses the siRNA strand that is incorporated into RISC depending on its structure.

A mild stimulation is sufficient for specific gene silencing in the entire organism, which denotes that the signal intensity can be amplified through RNAi silencing mechanism. This mechanism is termed as transitivity.

Simple interfering RNAs bearing additional (upstream/downstream) target site homology sequence sused to demolish the target. In this mechanism, the role of RdRP is vital. In RNAi, derivation of siRNA denotes the two fates: it can be transformed into holo-RISC that can be utilized for destruction of targeted mRNA, or may assist the amplification process by secondary siRNA generation.

Credit: nature video/Youtube.com

Uses of RNAi

Simple interfering RNA produced through the RNAi mechanism acts as an intracellular protection mechanism against invading viruses. In the laboratory, researchers utilize this method to block protein synthesis from target genes. It is simple and easy to synthesize and design a siRNA that binds to complementary mRNA.

Researchers are currently using  siRNA to develop therapies for human diseases that could be prevented by gene silencing. The mechanism of RNAi also acts as a powerful tool for biochemical studies to identify protein functions in various diseases.

Further Reading

• All MicroRNA Content
• What is MicroRNA?
• MicroRNA Nomenclature
• MicroRNA Biogenesis
• MicroRNA Cellular Functions