How RNA Interference Controls Gene Expression in Living Cells
RNA Interference Definition
The process within which RNA molecules inhibit the organic phenomenon by neutralizing the targeted mRNA molecules is called RNA interference.
The answer to the question of what is RNA Interference is that it is an evolutionarily conserved mechanism triggered by double-stranded RNA that uses the gene’s DNA sequence to show it off. This process is thought of as gene silencing. It is a gene regulatory mechanism that limits the amount of transcript in two ways. This process was discovered by two American scientists Craig C and Andrew Z.
1. Suppressing transcription
2. Degrading the RNA produced
RNA Interference Applications
The progress of RNA interference mechanisms has led to applications of this robust process in studies. Its RNA Interference Applications are as follows:
Gene Knockdown
RNA interference is usually accustomed to study the functions of genes in cell culture and model organisms. This mechanism is employed to scale back the expression of the targeted gene.
Functional Genomics
This technique is employed for gene mapping and annotation in plants. It has used for the studies in wheat bread.
Applications in Medicine
With the invention of synthetically made small interfering RNA, it became possible to silence the particular gene sequences rather than silencing the whole gene. Since then, RNAi has accustomed to target specific gene sequences that will cause cancer. It can even be accustomed to treat bacterial diseases, viruses, parasites, relieve pain, and also modulate sleep.
RNA Interference Steps
RNA interference (RNAi) is the biological mechanism by which small interfering RNA (siRNA) induces gene silencing through targeting complementary mRNA for degradation. This process is revolutionizing the way researchers study gene function. Its steps are as follows:
Step 1. Obtain Effective siRNAs
It is crucial to obtain gene silencing, potent and specific. Additionally, good experimental design dictates that a minimum of two effective siRNAs be employed in the experiment to substantiate that the observed effects result from flattening the gene of interest.
Step 2. siRNA Delivery to Maximize Gene Knockdown and Minimize Toxicity Optimization
Efficient, reproducible siRNA delivery is crucial for successful RNAi experiments. The first effective siRNA delivery protocol provides good gene knockdown while maintaining an appropriate level of cell viability. Negative control siRNAs are needed to identify potential non-specific effects on natural phenomena caused by introducing any siRNA.
Step 3. Test siRNA Silencing Efficiency
Because siRNAs exert their effects at the mRNA level, the single and most sensitive assay for siRNA validation relies on real-time RT-PCR to measure target transcript levels in cells transfected with gene-specific siRNAs versus negative control siRNAs.
Step 4. Examine Biological Impact of Silencing Target Gene
Assays that measure the results of gene silencing include morphological, enzymatic, biochemical, and immunological assays. siRNAs affect target mRNA levels, but phenotypic changes are usually due to the reduction of protein levels. siRNA-induced silencing at the protein level is typically measured by western blotting to correlate the observed phenotype with the quantity of knockdown induced
RNA Interference Processing
In the appropriate cell type and at the proper developmental stage, RNA (RNA) polymerase transcribes an RNA copy of a gene, the primary transcript. However, the primary transcript may contain more nucleotides than are needed to create the intended protein. Additionally, the primary transcript is prone to breakdown by RNA-degrading enzymes. Before the primary transcript is accustomed to guiding protein synthesis, it must be processed into a mature transcript, called messenger RNA (mRNA). It could be genuine in eukaryotic cells.
On an RNA molecule, the top formed earliest is understood because the 5′ (5-prime) end, whereas the trailing end, is that the 3′ end. The terms of the first transcript are particularly prone to a category of degradative enzymes called exonucleases. The CAP uses an unusual linkage between nucleotides. Exonucleases don’t recognize this unique structure and so cannot remove the CAP. Since exonucleases work only from an end, if the CAP nucleotide can’t be removed, the complete 5′ end of the mRNA is protected. The 5′ CAP also aids in transport out of the nucleus and helps bind the mRNA to the ribosome.
To protect the 3′ end against degradative exonucleases, a poly-A tail x added by a poly-A polymerase. Poly-A may be a chain of adenine nucleotides, 100 to 2 hundred units long. The poly-A tail has typical bonds that are prone to degradation by exonucleases. Still, it doesn’t have any protein-coding function, so it doesn’t particularly matter if a number of the A residues degraded. It takes quite some time for the poly-A tail to be lost entirely, and through now, the protein-coding portion of the mRNA remains intact. Without the poly-A tail, the exonucleases would rapidly degrade into the protein-coding part of the mRNA. An exception to the poly-A strategy seen within the mRNA for histones, proteins that wrap desoxyribonucleic acid (DNA) into chromosomes. Rather than poly-A, histone mRNA uses a far smaller structure that’s regulated by factors present during DNA synthesis.
FAQs on RNA Interference Explained: Definition, Steps, and Applications
1. What is RNA Interference (RNAi)?
RNA Interference, or RNAi, is a natural biological process in eukaryotic cells where small RNA molecules inhibit gene expression or translation. It works by neutralizing specific targeted messenger RNA (mRNA) molecules, effectively silencing the gene that produced them. This mechanism is a key cellular defence against viruses and transposable elements and also plays a role in regulating gene expression.
2. What are the main steps in the RNA interference mechanism?
The RNA interference pathway follows a precise sequence of events:
- Initiation: The process begins when a long double-stranded RNA (dsRNA) molecule enters the cell.
- Processing: An enzyme called Dicer recognizes and cleaves the long dsRNA into smaller, 21-23 nucleotide fragments. These fragments are known as small interfering RNAs (siRNAs).
- RISC Loading: The siRNA duplex is loaded into a protein complex called the RNA-Induced Silencing Complex (RISC). One strand of the siRNA (the passenger strand) is discarded, while the other (the guide strand) remains.
- Targeting and Cleavage: The guide strand within the activated RISC directs the complex to a complementary mRNA molecule. The Argonaute protein, a key component of RISC, then cleaves the target mRNA, preventing it from being translated into a protein.
3. How is RNA interference used in plant biotechnology to create pest-resistant crops?
RNA interference is a powerful tool in agricultural biotechnology. For example, to make a tobacco plant resistant to the nematode Meloidegyne incognita, scientists introduce nematode-specific genes into the plant. The plant then produces double-stranded RNA corresponding to these genes. When the nematode feeds on the plant roots, it ingests this dsRNA. Inside the nematode, the dsRNA triggers the RNAi pathway, silencing essential nematode genes and ultimately killing the pest, thereby protecting the plant.
4. What is the difference between siRNA and miRNA in the context of gene silencing?
While both are small RNA molecules involved in gene silencing, siRNA (small interfering RNA) and miRNA (microRNA) have key differences:
- Origin: siRNAs typically originate from exogenous sources like viral dsRNA or experimentally introduced dsRNA. miRNAs are endogenously encoded in the genome and are processed from precursor hairpin structures.
- Complementarity: siRNAs usually have perfect complementarity to their target mRNA, leading to direct cleavage and degradation of the mRNA.
- Mechanism: miRNAs often have imperfect complementarity, especially in animals, which typically leads to the repression of translation rather than immediate cleavage of the mRNA.
5. Why is double-stranded RNA (dsRNA) the primary trigger for the RNAi defence mechanism?
Double-stranded RNA is the primary trigger because its presence in the cytoplasm is a strong indicator of an anomaly. Most cellular RNA is single-stranded. The presence of long dsRNA is a hallmark of replication for many viruses or the activity of transposable elements (jumping genes). Therefore, cells have evolved the RNAi pathway as an ancient and highly specific defence system to recognize and destroy this foreign or aberrant genetic material, protecting the integrity of the host's genome.
6. What are the key protein components essential for the RNAi pathway to function?
Two main types of proteins are critical for RNA interference:
- Dicer: This is an RNase III-type enzyme responsible for recognizing long dsRNA and cutting it into short siRNA or miRNA fragments. It acts as the initiator of the pathway.
- Argonaute (Ago) proteins: These are the core components of the RISC complex. An Argonaute protein binds to the small RNA guide strand and uses it to find the target mRNA. It possesses the catalytic activity (slicer activity) that cleaves the target mRNA, executing the final step of gene silencing.
7. Besides agriculture, what are some potential applications of RNA interference in medicine?
The ability of RNAi to silence specific genes makes it a promising therapeutic strategy. Potential medical applications include:
- Antiviral Therapy: Developing RNAi-based drugs that target and destroy the RNA genomes of viruses like HIV, Hepatitis, and Influenza.
- Cancer Treatment: Silencing oncogenes (genes that promote cancer) or genes that confer resistance to chemotherapy, making treatments more effective.
- Genetic Disorders: Targeting and silencing mutated genes that cause hereditary diseases like Huntington's disease or certain types of macular degeneration.
