Molecular Biology Techniques Rna Interference

RNA interference, also known as RNA mediated interference or in short RNAi, is a mechanism of regulation of gene expression by certain RNA molecules such as miRNAs (micro RNAs), siRNAs (short interfering RNA/silencing RNA) etc. MicroRNAs are small single stranded RNA molecules of about 20-24 nucleotides in length and regulate the gene expression (usually silencing) of other genes. These RNAs are first formed from the noncoding RNA genes, in the form of long primary transcripts (pri-miRNA) that are processed to short nucleotide stem loop structures of about 70 nucleotides called pre-miRNA by microprocessor protein complexes (nuclease Drosha + Pasha) in the nucleus. These pre-miRNA molecules are then processed by endonuclease dicer (RNAse III ribonuclease) into a short lived miRNA:miRNA duplex, which is picked up the by the RISC (RNA induced silencing complex; degrades one strand from the miRNA duplex) to form highly specific miRISC complex that can down regulate the gene expression by two different mechanisms; either miRISC degrades the mRNA (if the miRNA and its corresponding mRNA have perfect complementarity) or repression of translation (if the miRNA and its corresponding mRNA have partial complementarity).
SiRNAs are short, double stranded RNA molecules that range from 20-25 nucleotides in length, involved in RNA interference pathway, where they interfere with the expression of some specific genes. The typical structure of these SiRNAs shows a 2 nucleotide overhang at the 3′ end that is thought to be generated by the dicer proteins while processing the long double stranded RNA molecules into these short double stranded siRNA molecules. The difference between the siRNAs and the miRNAs is that the former molecules are double stranded unlike those latter molecules which are single stranded.

Role of RNA interference in regulation of biological phenomenon:

The mechanism of RNA interference has been observed to play a vital role in many biological phenomenons such as maintenance of genome structure and organization, repression of gene expression, immunity (genome defense mechanism) etc.
RNA interference in genome maintenance: In many eukaryotic systems, the components of RNA interference pathway are used for the maintenance of genome struncture and organization. In Schizosaccharomyces pombe, it was observed that the RNAi machinery is essential for the heterochromatin formation at the mating type locus and for the silencing of the centromeric repeats, thus indicating that the RNA interference is an endogenous mechanism that is important for the chromosome stability. The heterochromatinization and histone modifications may also repress the gene expression by a phenomenon called pre-transcriptional downregulation of gene expression and the mechanism is termed as RNA induced transcriptional silencing (RITS). RITS is brought about by a complex of proteins know an RITS complex. In fission yeast, the RITS complex comprises of argonaute, Chp1 (a chromodomain protein) and Tas3 (function of this protien not yet known). Argonaute and RNA dependent RNA polymerase (RdRP) are required for the induction and spread of the heterochromatic regions and the deletion of these genes in Saccharomyces pombe negatively affect the histone methylation and the formation of centromere, thereby resulting in a slow or stalled anaphase during the cell division. It is still to be found by what mechanisms the RITS complex induces the formation of heterochromatin and its organization.
In Tetrahymena thermophila, an RNA interference related mechanism has been implicated, where the M-element small RNAs (sRNAs) are believed to be involved in targetting of the germline-limited sequences for chromatin modification and eventually for the DNA rearrangement. Thus the homologous sRNAs serve as mediators to communicate sequence-specific information between the parental and developing genomes, thereby regulating the genome-wide DNA rearrangement.
RNA interference in Immunity: RNA interference forms an important component of immune response to viruses and foreign genetic material. In plants this mechanism is especially important, as it prevents the self-propagation of transposons. In Arabidopsis thaliana, multiple dicer homologs are expressed in order to react differently with the different types of viruses. This phenomenon attributes an immune response memory to the plant and allows the entire plant to respond to a virus after an initial localized encounter. It has also been observed that some plants express the endogenous siRNAs when a bacterial infection is encountered and these RNA molecules are highly specific as far as the bacterial strains are concerned.
Some animals have also been shown to produce RNA molecules that interfere with the viral response. In Drosophila, the RNA interference establishes an antiviral innate immunity against its pathogens such as Drosophila X virus. In C. elegans, the upregulation of argonaute proteins takes place in a response to viruses and worms, which eventually over-expresses the components of the RNAi pathway in order to attain resistance to these infections. In mammals, little is known about the role of RNA interference in the innate immunity.

RNA interference in gene regulation and development:
miRNAs that are expressed endogenously form an important component of translational repression and in the regulation of development, such as the morphogenetic timing, maintenance of different stages of stem cells (undifferentiated or incompletely differentiated) etc. The role of RNA interference in the downregulation of the gene expression was first discovered in C. elegans. In plants this mechanism was first described in Arabidopsis, when the JAW microRNA was shown to be involved in the regulation of several genes (majority of these genes were transcription factors) that control plant shape. In humans and many other organisms, miRNAs have also not only been implicated in the formation of tumors but also in the cell cycle-dysregulation, thus showing that miRNAs may also function as both oncogenes and tumor supressors.

Practical applications of RNA interfernce:
The concept of RNA interference can be exploited to understand the possible function of any desired genes whose function is not known. A double stranded RNA can be synthesized that shows complementarity with the gene of interest and introduced into either the cells (cultured cells) or into an organism. This introduced dsRNA is recognized by the host cell as a foreign (exogenous) genetic material and activates the RNAi pathway, thereby repressing the expression of corresponding gene transcripts and allowing us to possibly understand the gene function. Apart from gene knock out studies, RNA interference can also be used for silencing human disease genes, engineering plants for desired effect such as delaying ripening of tomatos for long shelf life etc.
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