[Biology Class Notes] on RNA Interference Pdf for Exam

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:

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.

This technique is employed for gene mapping and annotation in plants. It has used for the studies in wheat bread.

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.