RNA interference

http://en.wikipedia.org/wiki/RNA_interference

Definition:RNA interference (RNAi) is a mechanism where the presence of certain fragments of ds RNAinterferes with the expression of a particular gene which shares a homologous sequencewith this dsRNA.

Before RNAi was well characterized, it was called by other names, including post transcriptionalgene silencing (TGS) and transgene silencing. Only after these phenomena were characterized atthe molecular level was it obvious that they were the same phenomenon.

The use of RNA to reduce expression in plants has been a common procedure for many years.Single-stranded antisense RNA was introduced into plant cells that hybridized to the cognate,single-stranded, sense messenger RNA. While scientists first believed that the resulting dsRNAhelix could not be translated into a protein, it is now clear that the dsRNA triggered theRNAi response. The use of dsRNA became more widespread after the discovery of theRNAi machinery, first in petunias and later in roundworms (C. elegans).

RNAi is a specific process, known as the RNA interference machinery. It appears that the machinery,once it finds a double-stranded RNA molecule, cuts it up with an endonuclease (Dicer), separatesthe two strands, and then proceeds to destroy other single-stranded RNA molecules that arecomplementary to one of those sequences. dsRNAs direct the creation of small interfering RNAs(siRNAs) which target RNA-degrading enzymes (RNAses) to destroy transcripts complementaryto the siRNAs.

The life cycle and replication of many RNA viruses involves a double-stranded RNA stage,so it is likely that part of the RNA interference machinery evolved as a defense against these viruses.

The machinery is however also used by the cell itself to regulate gene activity:certain parts of the genome are transcribed into microRNA, short RNA molecules that fold backon themselves in a hairpin shape to create a double strand. When the RNA interference machinerydetects these double strands, it will also destroy all mRNAs that match the microRNA,thus preventing their translation and lowering the activity of many other genes

This mechanism was first shown in the "JAW microRNA" of Arabidopsis; it is involved in theregulation of several genes that control the plant's shape. The mechanism has also been showin many other eukaryotes; by now, more than 200 microRNAs have been detected in humans.

RNAi has been linked to various cellular processes, including the formation of centromeric structure and gene regulation, through microRNAs and heterochromatin formatio

siRNA (small interfering RNA)http://en.wikipedia.org/wiki/Small_interfering_RNA

Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a classof 20-25 nucleotide-long RNA molecules that interfere with the expression of genes.They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme Dicer.They can also be exogenously (artificially) introduced by investigators to bring about th knockdown of a particular gene.

siRNA's have a well defined structure.Briefly, this is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt overhangson either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.

Transfection of an exogenous siRNA is problematic, since it is only transient, and thedsRNA structure cannot easily be permanently maintained.One way of overcoming these problems is to modify the siRNA in such a way as to allow itto be expressed by an appropriate vector, e.g. a plasmid. This is done by the introductionof a loop between the two strands, thus producing a single transcript, which can be processedinto a functional siRNA.

This transcription cassette usually uses an RNA polymerase III promoter, which direct thetranscription of small nuclear RNA's, such as U6 or H1. It is assumed (although not knownfor certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer.

siRNA

Introduction of too much siRNA can result in non-specific events due to activation of the interferonpathway. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR,although retinoic acid inducible Gene I (RIG-I may also be involvedOne method of reducing the non-specific effects is by turning the shRNA into a micro RNA.Micro RNA's are naturally occurring, and, as such, tolerated better by the cell.By engineering an siRNA sequence into an miRNA structure, non-specific effects canpotentially be eliminated.

The mediators of RNA interference are 21- and 23-nucleotide small interfering RNAs (siRNA).siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) thatguides the small dsRNAs to its homologous mRNA target.

Consequently, RISC cuts the mRNA approximately in the middle of the region pairedwith the antisense siRNA, after which the mRNA is further degraded.http://www.qbiogene.com/products/transfection/app-sirna.shtml

miRNA

miRNA (micro-RNA)http://en.wikipedia.org/wiki/MiRNA

A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long.It is thought to regulate the expression of other genes.

miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequenceincludes the miRNA sequence and an approximate reverse complement. When this DNA sequence istranscribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement basepair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA.The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to releasethe mature miRNA.In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takesplace in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.

miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.The DNA sequence that codes for an miRNA gene is longer than the miRNA itself.

This DNA sequence includes the miRNA sequence and an approximate reverse complement.

The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,a carrier protein.

The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to releasethe mature miRNA.

In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takesplace in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.

The function of miRNAs appears to be in gene regulationFor that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs).

Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs areusually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA theinhibits protein translation, but sometimes facilitates cleavage of the mRNA.This is thought to be the primary mode of action of plant mIRNAs.In such cases, the formation of the double-stranded RNA through the binding of the miRNAtriggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi),though in other cases it is believed that the miRNA complex blocks the protein translationmachinery or otherwise prevents protein translation without causing the mRNA to be degraded.miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs.

Gene silencing in plants occurs at both transcriptional (TGS) and posttranscriptional (PTGS) levels.Double stranded RNA (dsRNA), which is processed to short interfering RNAs (siRNA) of 21-24 nucleotides, plays a key role as an inducer in both silencing processes.

The siRNAs corresponding to promoter sequences direct the silencing machinery to bloctranscription of homologous promoters (TGS), whereas

those corresponding to transcribed sequences direct the silencing machinery to degradehomologous RNAs (PTGS). TGS accompanied by chromatin methylation and remodeling;and PTGS associated with RNA degradation can neutralize endogenous and exogenouinvaders such as transgene, viruses and transposon.