“A sequence-specific mRNA degradation is governed by the smaller dsRNA mediated process called the RNAi or RNA interference.”


 RNA interference often abbreviated as RNAi, is a process in which the exogenous and endogenous RNA degraded, which consequence in gene silencing. 

In the year 1993, Andrew Fire and Craig Mello postulated the mechanism of RNA interference by introducing the exogenous dsRNA into the C. elegansBoth were awarded Nobel prize in the category of physiology or medicine in 2006. 

In the year, 2013, fortune magazine announces the discovery of RNAi as “Biotech’s Billion Dollar Breakthrough”. 

The RNAi is a pathway of the innate immune system of us which protects us from the attacks of exogenous dsRNA. 

RNA is a genetic material of some viruses called the retroviruses other than that only dsDNA is genetic material in all prokaryotes as well as eukaryotes. 

Thus, the presence of the dsRNA in our cell is a sign of danger for us. Our cells have developed a natural mechanism protecting against the exogenous dsRNA called the “RNA interference”. 

In the present article, we will focus on the mechanism and applications of RNAi.

Key topics: 

  • The process of RNAi
  • RNAi as a genetic tool
    • characteristics of si/shRNA for RNAi
    • In vitro process of RNAi
  • Applications of RNAi
  • Conclusion

The process of RNAi is governed by smaller microRNAs that degrades the mRNA and does do gene silencing. 

We have already discussed the process of RNAi in our previous article. Read it here: microRNA (miRNA) and Gene Regulation.

The process of RNAi:

Briefly, the process is as followed, 

In the very first step, the primary miRNA is transcribed from a gene by the RNA pol II. 

The protein called drosha process the Pri-miRNA and forms the precursor miRNA transported to the cytoplasm using the protein exportin 5. 

At cytoplasm, the enzyme dicer cleaves the pre-miRNA at the loop-stem junction (in case of shRNA) and creates a double-stranded microRNA, with two nucleotide overhang at 3′ end. 

One of the dsRNA strands is called the guided strand, a complementary strand to our target mRNA and another strand is a passenger strand. 

The small dsRNA is now loaded in the RISC, RNA induced silencing complex. 

In the very next step, the passenger strand is degraded and the guided strand along with the RISC loaded on the target mRNA. 

The RISC is a complex of many proteins, Argonaute protein is one of them. The Ago2 protein finds the complementary bases of our guided strand on the 3’ end of the mRNA and cleaves it. See the structure of RISC: 

The structure of Argonaut protein and RISC.

This Ago mediated cleaving consequently inhibits the expression of a protein that encodes.

In another mechanism, if the RISC fails to find the complementary strand, still, it binds to the target mRNA and aborts the transcription which ultimately fails to do protein formation. 

Thus in either mechanism, a gene can not be expressed. 

The process of gene silencing by miRNA.

The entire process of miRNA mediated RNA interference.

Besides their role in gene silencing, the RNAi mechanism also facilitates protection against accumulation of transposons and other repetitive DNA sequences and thus protects against genomic instability. 

The RNA interference is naturally governed by the microRNA, abbreviated as miRNA, that is a transcript of a gene but can not undergo translation. 

The innate immune system component RNAi, not only involved in the gene regulation but it is also functioning in cellular defence against RNA viruses and transposon suppressing. 

As we discussed above, the retroviruses have the RNA as their genetic material, rapidly replicates in the host and causes some disease conditions.

For example the influenza virus, HIV and rabies virus ( rhabdovirus). 

The eukaryotic antiviral RNAi pathway activated once the RNA of the exogenous virus is inserted into the cell. 

A short segment of the RNA complementary to the viral RNA is synthesised which later binds to the viral RNA and processed it via RNA interference. 

The transposons often called a “jumping genes” can produce copies of identical DNA segments within different parts of a genome and produces repetitive DNA sequences. 

These unwanted DNA segments sometimes affect gene expression and thus giving rise cancer-like conditions. 

The natural RNA interference finds the activated transposons and processed them during post-translational gene silencing and prevent the unwanted accumulation of repetitive DNA sequences. 

The findings of Zamore DP et al suggest that the RNAi pathway required ATP as an energy source to perform the function of post-transcriptional gene silencing. 

Because of the tremendous applications of RNA interference mechanism in gene silencing and gene expression regulation, it is one of the popular experimental molecular tool used in the gene expression studies nowadays. 

Hence, it is named as “breakthrough of the year” when the mechanism was discovered. 

RNAi as a genetic tool…

Alike the siRNA of a cell, synthetic dsRNA complementary to our target mRNA is designed and introduced in the cell line using the expression vectors. 

Once it is properly introduced in a cell, the remaining process is performed by the cell RNA interference mechanism itself. 

The exogenous dsRNA is identified by the dicer and cleaved into 21 to 23nts fragments of dsRNA. 

Dicer process it and transfers it to cytoplasmic RISC, at which the Ago2 protein of the RISC binds to the siRNA fragments. 

The passenger strand of the siRNA fragments which is similar to the mRNA is discarded and another strand called the guided strand remains in the complex. 

After that, the RISC migrates it to the complementary mRNA binds to it and degrades the mRNA. 

The mRNA can not be translated into protein result in the reduced gene expression. 

The siRNA is a main ingredient of the in vitro RNAi experiments, which is generally 21 nucleotides in length called 21mer. 

The 21mer siRNA is more specific and work excellently experimentally, however, the recent discoveries suggest that instead of 21mer, the 27mer siRNA operates more preciously. 

The 27mer is cleaved properly by the dicer and produces the 2 nucleotide overhang at the 3’ end of it. 

This siRNA looks more similar to the endogenous miRNA. Read mour article on siRNA: siRNA (Small Interfering RNA): Structure And Function.

Another molecule called the shRNA, or short hairpin RNA is also used in in vitro RNA interference experiments. The structure of siRNA and shRNA is shown in the figure below,

The structure of shRNA and siRNA

The structure of shRNA and siRNA.

By increasing the sequence specificity and subsequently reducing the ability to cross-hybridization with chemical modification can increase the effectiveness as well as the specificity of the siRNA in the RNAi process. 

The selected synthetic nucleic acid (siRNA or shRNA) must have several characteristics to trigger RNAi responds more appropriately. 

Characteristics of selected sh/siRNA:

Chemical modification: In the siRNA mediated RNA interference there is no need to do the chemical modification, however, to increase more specificity and to reduce the off-target activity, siRNA must be chemically modified. 

The siRNA must have the 5’ phosphate group hence it is necessary not to remove it during the experiment. 

Furthermore, the addition of dTdT nucleotides at the 3’ end should increase the RISC loading of the guided strand, for that one required chemical modifications to do so. 

However, the modified siRNA must be tested first, before introduction otherwise, the nuclease misses the dsRNA degradation. 

The siRNA used in the RNAi must be thermodynamically favourable. 

The siRNA must have low melting temperature and duplex stability towards the 5’ end of the guided strand and high melting temperature and high duplex stability towards the passenger strand. 

Thus, the passenger strand can be removed and consequently degraded properly. 

Furthermore, adding the mismatch at the 5’ end of the guided strand can increase the functionality of the siRNA (which reduce the stability). 

In addition to this, 

The synthetic double-stranded RNA does not have more than four identical nucleotides adjacent to each other and moderate to low GC content. Typically, 30 to 45% GC content is appropriate for RNAi. 

Tips: 

Design two or three siRNA target for one particular gene and make sure that each one triggers the RNA interference particularly for that gene which gives similar results in each experiment. 

In the next step of the siRNA mediated RNA interference, it is necessary to check the off-target effect of the exogenous NA duplex. 

For that, an appropriate amount of siRNA dose is needed. 

Deciding a dose for RNAi triggering is crucial, start with the lower dose and subsequently increase the dose of siRNA. 

Chose the concentration which gives effective results and has lower off-targets. 

The higher dose of the exogenous dsRNA can disturb the function of dicer as well as the RISC and results in off-target activity. 

In vitro process of RNAi:

Different steps in RNAi experiment design are shown in the figure below,

The in vitro process of RNAi

The in vitro process of RNAi: (1) selection of target gene for gene silencing, (2) designing the si/shRNA specific to the target gene, (3) selecting a plasmid or vector (4) introducing the dsRNA to cells and (5) gene expression assay.

What is off-target? 

The off-target effect in the RNA interference mechanism occurs when the dsRNA guides degradation of non-target mRNA consequently results in the gene silencing of the undesirable gene. 

Another synthesis RNA molecule is often used in the RNAi experiments is known as shRNA

The shRNA, short hairpin RNA is an artificially synthesised single-stranded RNA loop created through heat annealing. 

Ultimately it creates a fold of dsRNA having a 2 nucleotide overhang. 

Cleaving through the dicer, it resembled like the endogenous miRNA and processed by the RISC for gene silencing. 

The entire mechanism of shRNA mediated gene silencing is explained in our previous article, read it here: What Is shRNA (Short-hairpin RNA)?

Related article:

  1. Reverse transcription PCR: Principle, Procedure, Applications, Advantages and Disadvantages.
  2. Gene Therapy: Types, Vectors [Viral and Non-Viral], Process, Applications and Limitations.

Applications of RNAi: 

The use of the artificial dsRNA molecules induces RNA interference response of a cell and results in the regulation of the gene expression. 

Thus the Artificially induced RNAi has several tremendous application in the clinical, therapeutic and other research fields.

It is widely used now in gene knockout studies. 

Also, it is implemented in functional genomics research and studies. 

Therapeutically, it is now used against viral infections, cancer and neurological diseases, researchers aiming to use it as a safer therapy to cure diseases. 

RNAi therapies can also be used in personalised medicines and targetted-gene therapy.

In recent years, RNAi technology has greatly emerged in plant research and crop improvements. 

Scientists are now using the RNAi and antisense RNA technology in crop improvement. 

Novel plant traits and disease-resistant species of plants are being developed using the present technology. 

Moreover, it is also used in pest control and yield improvement. 

Flvr Savr tomato, decaffeinated coffee and nicotine-free tobacco are some of the best examples of plant species developed using RNAi technology. 

The RNAi is also used for disease and pathogen resistance, development of male sterility and functional genomic studies in plants. 

Virus resistance plant species against Banana Bract Mosaic Virus, Rice Tungro Bacilliform Virus, Tobacco Mosaic Virus and Cucumber Mosaic Virus are developed by using the antiviral RNA silencing through RNAi. 

Artificially engineered dsRNA complementary to the viral RNA is introduced into the plant genome which mimics like the natural si/miRNA and destroys viral dsRNA every time it attacks. 

Conclusion:

Preventing cancer is one of the tedious work for scientists, although the promising outcomes of the RNAi like technologies raise hope for it. Still, scientists have to do a lot of research to establish it as a successful therapy for preventing cancer.

In addition to this, infectious diseases can be prevented more powerfully using RNAi.

In the crop improvement, the RNAi and antisense RNA technology have tremendous potential to create new variation in plant species for increasing yield and resistance against pathogens.

Sources: