Table of Contents -
RNA interference
RNA interference was first studied by Andrew Fire & Craig Mello in C.elegans in the year 1998.
RNA interference is also abbreviated as RNAi.
It is also called PTGS i.e Post transcriptional gene silencing or also called as transgene silencing or also called as quelling.
It is observed in all Eukaryotes from yeast to mammals.
Types of RNA interference
A) siRNA mediated RNA interference
siRNA mediated interference :- “OCCUR IN CYTOPLASM ONLY”.
Steps of siRNA mediated interference
1) Processing of dsRNA to siRNA duplex with 2nt overhang at 3’ end by Dicer enzyme.
2) Dicer and dsRNA binding protein form RISC [RNA inducing silencing complex] complex and they load siRNA duplex in RISC.
3) Active component of RISC i.e Argonaute proteins (Endonuclease) cleave target mRNA complementary to bound siRNA or guide strand.
[Guide strand of siRNA bind complementarily to target mRNA whereas Anti-guide or Passenger strand of siRNA gets degraded during RISC Activation]
About Dicer Enzyme :- Molecular weight is 200Kda, and belong to Rnase-III Family Enzyme.
Dicer Has Multidomains :-
i)ATPase/RNA Helicase domain
ii)Catalytic RNase III domain
iii)dsRNA binding domain
Dicer Enzyme Image Retrieved From Molecular Biology Third Edition Book by David Clark, Nanette Pazdernik, Michelle McGehee
B) miRNA mediated RNA interference
miRNA mediated interference :- Half process occur in Nucleus and Half in Cytoplasm.
Steps of miRNA mediated interference which occur in Nucleus
1) RNA Pol-II and RNA Pol-III transcribe some pri-mRNAs.
2) pri-mRNAs folds into hair pin structure.
3) Drosha and dsRNA binding protein processes pri-mRNA into hairpin RNAs (70-100 nulceotides) and forms pre-miRNA.
4) Pre-miRNA is exported from nucleus to cytoplasm by Exportin-5 protein. It is Ran-GTP dependent export thus utilize GTP.
Steps of miRNA mediated interference which occur in Cytoplasm
1) Dicer Enzyme processes Pre-miRNA to miRNA duplex and load into RISC. RISC brings or directs miRNA toward target mRNA
2) miRNA’s Guide strand bind to 3’ UTR of target mRNA.
3)Argonaute protein (Argonaute 2 protein plays major role in siRNA and miRNA interference) cleave target mRNA. Note that, Passenger strand is also degraded by Argonaute protein.
About Argonaute protein :- It has 2 RNA Binding domains
i) PAZ Domain :- Bind single stranded 3’ end of mature miRNA
ii) PIWI Domain :- Resemble RNase H
Characteristics of Plant miRNA :-
1) Perfect or near perfect pairing with target mRNA.
2) Single Enzyme DCL-1 play role of both Drosha and Dicer.
3) Before pre-miRNA duplex transported from nucleus, its 3’ overhangs are methylated by S-adenosylmethionine dependent methyltransferase enzyme called HEN-1.
Characteristic feature of Animal miRNA :-
1) Only recognize 6-8 nucleotides of target mRNA.
Points about miRNAs :-
1) miRNA can have multiple different mRNA targets since miRNA show incomplete complementary binding with mRNA.
2) Pre-miRNAs also derived from introns called mirtrons.
3) miRNA biogenesis differ in plants and animals in steps of export and nuclear processing.
4) dsRNA binding protein in Drosophila is Pasha, and DGCR8 in Mammals [Drosha and DGCR8 complex is called Microprocessor complex which processes pri-miRNA to pre-miRNA].
C) piRNA mediated RNA interference
piRNA (Piwi interacting RNAs) :- Expressed in Germ cells and regulate transposon activity in germ cells. piRNA is involved in the degradation of transposon mRNA or transposon gene silencing
Transposons or Jumping genes or mobile genetic elements have the ability to integrate themselves randomly anywhere into the genome. This can result in mutations in the gene and disrupt the coding sequence of the gene which can be fatal for an organism. Changes in genome is also inheritable which can affect future generation. Thus, there occurs a need to control random integration of transposon. Although, it is important to note that, transposon can still show transposition. piRNA mediated RNA interference cannot prevent it completely
piRNAs are mostly in antisense (3′ –> 5′ orientation) but small fraction of piRNAs are in sense (5’ –> 3’ orientation).
piRNA has 2’-O-methylated at 3’ end unlike miRNA but similar to siRNA.
Steps in piRNA mediated RNA interference
1) piRNA clusters are transcribed into Primary piRNAs in the nucleus
[Single stranded piRNA precursor formed without dicing step]
2) Primary piRNAs are then exported from nucleus to cytoplasm
3) Mitochondria which are present in cytoplasm have zucchini endonuclease which cause cleavage and generate proper 5′ end of primary piRNAs. Nibbler exonuclease is also present in the cytoplasm which trims the primary piRNAs in 3′ –> 5′ direction. These both processes results in the formation of Mature piRNA
4) Mature piRNAs are loaded onto Piwi or Aub proteins, a subfamily of Argonaute proteins [Ago 3 protein is involved in piRNA mediated RNA interference]
5) Piwi-piRNA complexes scan the genome for transposons and bind complementarily to them and catalyze the cleavage of transposon transcript or mRNA
6) Upon cleavage of transposon transcript, secondary piRNAs are produced which can participate in the degradation of additional transposon transcripts. This feedback loop is a characteristic feature of piRNA mediated RNA interference
7) IN NUCLEUS :- piRNA mediated RNA interference cause Transposon gene silencing by DNA methylation [Transcriptional gene silencing]
IN CYTOPLASM :- piRNA cause cleavage of transposon mRNA [Post-Transcriptional Gene Silencing]
Function of piRNA :-
1) Cleavage of transposon mRNA.
2) Chromatin modification at transposon loci.
Functions of RNA interference
1) RNA interference play an important role in defense against virus.
2) RNA interference play a crucial role in transposon regulation especially by piRNA mediated RNA interference. Transposons or Jumping genes or mobile genetic elements have the ability to integrate themselves randomly anywhere into the genome. This can result in mutations in the gene and disrupt the coding sequence of the gene which can be fatal for an organism. piRNA mediated RNA interference process can result in the degradation or cleavage of the transposon mRNA or it can also transposon gene silencing by epigenetic modification like DNA methylation.
3) RNA interference is widely used to silence a particular gene expression which may be harmful or disadvantageous for an organism. For example, if we need to silence a cancer causing gene expression in human, we can do it with the help of RNA interference technique.
- siRNA causes gene silencing by cleavage of target mRNA whereas miRNA causes gene silencing by inhibition of translation.
4) RNA interference can also be used to produce pest-resistant plants. Example – One tobacco plant was produced to provide resistance against pest or nematode Meloidogyne incognita using this technique.
5) RNA interference technique is also widely used to determine the function of novel or new gene. If we silence a particular gene, we can study what will be the effect upon gene silencing as compared to when the gene is active or functional. This is how, we can determine the function of a desired or new gene.
6) RNA interference is also used for gene knockout when conducting experiments for Recombinant DNA Technology and Genetic Engineering.
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