RNA interference

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RNA interference:

RNA interference Ajay Prakash Uniyal M.Sc Plant Science Central University of Punjab

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RNA interference is evolutionary conserved mechanism of gene regulation, it is sometimes called quelling. siRNA mediated RNAi. ( artificially or produced in vivo from dsRNA precursors) miRNA mediated RNAi.( Derived from precursor RNAs that are encoded by genes) A third class of short regulatory RNAs is the piwi -interaction RNAs ( piRNAs ), which are expressed predominantly in the germline and have features distinct from miRNAs Both siRNAs and miRNAs are generated from longer RNA molecules by the enzyme Dicer, an RNase III–like enzyme that recognizes and digests longer dsRNAs or the stem-loop structures formed by miRNA precursors

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Synthesis and function of miRNA miRNAs are encoded in the genome as segments of longer transcripts. The functional form of an miRNA is typically 21 or 22 nucleotides ( it can vary from 19 to 25 nucleotides). These short RNAs are generated by two RNA cleavage reactions from a longer RNA transcript ( called a pri miRNA , for “primary”) that carries a hairpin-shaped secondary structure . The first cleavage liberates the stem-loop, called the pre-miRNA; T he second generates the mature miRNA from the pre-miRNA. One of the first identified, and best-characterized, miRNAs is let-7, which regulates development at the larval-to-adult transition in the worm C. elegans

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The two cleavage reactions required to generate the miRNA from these primary transcripts are mediated by two distinct RNases. 1.Dicer. 2. Drosha . Drosha makes two cleavages that cut the stem-loop region of the RNA ( pre-miRNA ) out of the primary transcript RNA ( pri -miRNA). usually 65–70 nucleotides long. Drosha resides in the cell’s nucleus, and the Drosha -catalyzed cleavage event occurs in that cellular compartment The pre-miRNA generated by Drosha is 33 bp in length.

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The pre-miRNA liberated by Drosha is exported to the cytoplasm, where the second RNA cleavage reaction, performed by Dicer, takes place. As with Drosha , Dicer selects its cleavage sites using a measuring, rather than sequence-specific , mechanism . Cleavage by Dicer generates a suitable 21- to 22-nucleotide RNA for incorporation into RISC. The central component of RISC is a protein called Argonaute , which is in many cases an RNA-cleaving enzyme . The short dsRNA generated by Dicer is incorporated into RISC , where it is denatured to provide the guide strand and the passenger strand (which is discarded). The resulting RISC—called mature RISC— with its single-stranded guide RNA is now ready to recognize and slice the target mRNA . The bound guide RNA is base-paired to the target RNA, and the architecture of the complex is such that this binding positions the active site of the RNase domain appropriately to cleave the target RNA strand. Cleavage occurs nearly in the middle of the guide RNA–target RNA duplex, between the 10th and 11 th nucleotides from the 50 end of the guide RNA.

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Difference between miRNA of Plants and Animals In plants, single enzyme DCLQ present inside the nucleus fills the roles of both Drosha and Dicer, converting pri -miRNAs to mature miRNA. Before plant miRNA duplex is transported out of nucleus, its 3’ overhangs are methylated by S adenosyl methionine dependent methyltransferase called HEN1. Gene silencing by miRNA may occur by mRNA degradation or preventing mRNA from translation in animals but in plants its through degradation only.

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siRNA mediated RNAi In siRNA mediated RNAi pathway, ds RNAs by the DICER is processed into siRNA duplexes , comprised of 21 nucleotides long strands. The Nucleotides are overangs at 3’ end. Dicer and dsRNA binding protein then load the RNA duplex into RISC. Only one of the strands called guide strand directs the gene silencing. The other strand called passenger strand is degraded by RISC activation.

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Diseases assosciated with RNAi INHERITED DISEASE Mutation of miR-96 in the seed region of humans results in hereditary progressive hearing loss. Mutation of miR-184, causes in the seed region results in hereditary keratoconus and anterior polar cataract in humans. CANCER Chronic lymphocytic leukemia was the first reported human disease that occurs due to the miRNA deregulations. There are also many other cancer related problems that occurs due to miRNA deregulation. HEART DISEASES In murin heart miRNA plays an global role in the heart by regulating miRNA matuaration . miRNA also plays a role in cholesterol metabolism. Some miRNA moleculs acts as potential biomarkers for cardiovascular diseases.   NERVOUS DISORDER Deregulation of MicroRNA results in several nervous disorders.

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piRNAs are the third class of small regulatory RNAs (after siRNAs and miRNAs ); they arise from long , single-stranded transcripts of piRNA clusters in the genome, without the need for Dicer action . Piwi interacting RNAs. They are 2’0 methylated at their 3’ termini unlike miRNAs but similar to siRNAs. piRNAs direct cleavage of transposon mRNA or chromatin modification of transposon loci.

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