RNA Interference or RNAi
Introduction
RNA interference (RNAi) or Post-transcriptional gene silencing (PTGS) is a process of gene silencing or sequence-specific mRNA degradation. It is a phenomenon inside a cell (either eukaryotic or prokaryotic) that targets and selects the mRNA strand to cleave off and degrade it, so it will block the synthesis of functional unit protein.
The process of gene silencing provides resistance to endogenous and exogenous parasitic or pathogenic nucleic acids; moreover, it is also used for the regulation of gene expression, having its functional applications in therapeutics, genomics, and other research-related areas.
The process of gene silencing was discovered by Scientists Andrew Z. Fire and Craig C. Mello, early in the 1900s. Both scientists performed their work by successfully inhibiting the expression of sequence-specific genes in the cells of Nematodes for example; C. elegans (Caenorhabditis elegans).
Generally, RNA is a single-stranded molecule but it may be converted into a double-stranded (ds) molecule inside a body, it leads to the cleavage of the ds-RNA molecule by an enzyme known as RNase III DICER which produces small fragments of 20 – 25 base pairs of si-RNA or mi-RNA which leads to the silencing of a specific sequence of mRNA and no functional protein product is formed. This is known as RNA-mediated gene silencing.
The process of gene silencing is carried out when many other proteins are combined and form a complex known as RISC, for RISC recruitment there is a requirement of ATP which unwinds the short fragments of ds-RNA (siRNA or miRNA) into guide and passenger strands respectively. The passenger strand is degraded but the guide strand combines with Argonaute protein and is used to recognize the specific sequence of nucleotides on the mRNA and degrade it, no functional protein product is formed.
Mechanism of RNAi
The process of RNAi or gene silencing is mediated by the endogenous RNAi pathway having foreign DNA or viral ds-RNA molecule. This pathway is divided into two steps:
- The cleavage of ds-RNA molecules into Si-RNA, mi-RNA by the action of DICER.
- The degradation of a specific sequence of mRNA molecules by RISC recruitment to make it non-functional
When there is a ds-RNA molecule of a viral origin, an enzyme RNase III Dicer (which is a member of RISC) will initiate the pathway in such a way that the ds-RNA will be cleaved off into short fragments (21-23 nucleotide in length with 3’overhang) either Si-RNA or mi-RNA depending upon the origin having heterochromatin or transposons in case of Si-RNA or specific sequence of genes for mi-RNA.
The fragments formed by cleavage will be loaded into the RISC by using ATP as a precursor. The RISC will unwind the ds-RNA short fragments (si-RNA, mi-RNA) and lead to the production of two strands: the guide strand and the passenger strand. RISC degrades the passenger RNA strand because it might block the recognition of the specific nucleotide sequence which has to be silenced, while the guide strand is taken by the AGO protein.
In the case of si-RNA or mi-RNA, the AGO protein selects that guide strand that has a less thermodynamically stable 5’end. It will further bind to the particular sequence of nucleotides on mRNA molecules which has to be degraded and will block the synthesis of the protein, (RNA mediated gene silencing).
In the case of primary mi-RNA, it is processed first by nuclear RNase III Drosha enzyme and releases hairpin-like pre-mi-RNA fragments that are subsequently cut by cytoplasmic RNA III DICER to generate mi-RNA fragments.
Difference between si-RNA and mi-RNA mediated pathways
Mature mi-RNA are structurally similar to si-RNA as they are derived from the same ds-RNA but with few exceptions in primary mi-RNA processing as I already described above the processing of mi-RNA by the nuclear RNase III Drosha enzyme. Moreover, both si-RNA and mi-RNA have the same downstream machinery.
mi-RNA is a short fragment that is derived from cleavage of ds-RNA and it differs from that of si-RNA, as in animals mi-RNA has mostly incomplete base pairing which may mimic the sequence of targeted mRNA and binds to certain different mRNAs and block their activity by silencing the gene. On the contrary, si-RNA has almost perfect base pairing and cleaves of the targeted mRNA at a specific site.
DICER and RISC
Endoribonuclease DICER or RNase motif, an enzyme that is present in humans and is encoded by the DICER1 gene. It is a part of the RNase III family that cleaves the ds-RNA and si-RNA or mi-RNA almost 20-25 base pairs in length.
Its basic function is its involvement in the activation of RISC which is responsible for the silencing of sequence-specific mRNA molecules. The domains of DICER taking part in RNAi are PAZ (PIWI/Argonaute/Zwille) and helicase in the case of humans, and capable of being binding to the 2 nucleotides 3’ overhang of ds-RNA molecule and processing of long substrates respectively.
Whereas RISC is a combination of multiple proteins that form a complex as ribonucleoprotein and its primary motive is to silence a gene by degrading the mRNA at a specific site. The single-stranded RNA fragment acts as a guide for the RISC to recognize the mRNA at the targeted site while the other one is degraded.
The process of recognition and cleavage is done by a protein present in a complex known as Argonaute, which binds to the small non-coding RNA (si-RNA, mi-RNA, and Piwi-interacting RNA), and leads to gene silencing.
Importance of RNAi and limitations
RNAi is essential as we can silence/knockout a gene in a biological system without hampering the DNA molecule, in the case of humans we can remove a mutant gene that may lead to cancerous protein formation, moreover it has the majority of its applications in therapeutics, genomics and agriculture fields.
The limitations of RNAi include the long-term use of medication as the gene has been successfully being silenced but to prevent the formation of protein certain medicines have been used. Another limiting factor of RNAi is the development of resistance against certain mRNA molecules which not only affect the gene silencing but also impose certain other diseases that might lead to the death of a person.
Conclusion
RNAi is an essential phenomenon in certain respects like gene silencing, production of useful products in the fields of agriculture and therapeutics but if it acquires resistance or certain other factors may affect its functioning then it will be life-threatening.
Reference and Sources
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC309050/
- https://www.researchgate.net/publication/330002643_Gene_Silencing_The_Mechanism_to_Down_Regulate_the_Target_Gene
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048316/
- https://wikimili.com/en/RNA_interference
- http://web.mit.edu/beh.109/www/Module3/handouts/Handout%20Lect%201%20RNAi%20-%20www.ambion.com-techlib-hottopics-rnai.htm