RNA interference

1. RNA interference http://en.wikipedia.org/wiki/RNA_interferenceDefinition: RNA interference (RNAi) is a mechanism where the presence of certain fragments of ds RNA interferes with the expression of a particular gene which shares a homologous sequence with this dsRNA. Before RNAi was well characterized, it was called by other names, including post transcriptional gene silencing (TGS) and transgene silencing. Only after these phenomena were characterized at the 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 dsRNA helix could not be translated into a protein, it is now clear that the dsRNA triggered the RNAi response. The use of dsRNA became more widespread after the discovery of the RNAi machinery, first in petunias and later in roundworms (C. elegans).

2. 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), separates the two strands, and then proceeds to destroy other single-stranded RNA molecules that are complementary to one of those sequences. dsRNAs direct the creation of small interfering RNAs (siRNAs) which target RNA-degrading enzymes (RNAses) to destroy transcripts complementary to 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 back on themselves in a hairpin shape to create a double strand. When the RNA interference machinery detects 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 the regulation of several genes that control the plant's shape. The mechanism has also been show in 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

3. siRNA siRNA (small interfering RNA)http://en.wikipedia.org/wiki/Small_interfering_RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class of 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 overhangs on 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 the dsRNA structure cannot easily be permanently maintained. One way of overcoming these problems is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction of a loop between the two strands, thus producing a single transcript, which can be processed into a functional siRNA. This transcription cassette usually uses an RNA polymerase III promoter, which direct the transcription of small nuclear RNA's, such as U6 or H1. It is assumed (although not known for certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer.

4. Introduction of too much siRNA can result in non-specific events due to activation of the interferon pathway. 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 involved One 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 can potentially be eliminated.

8. 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) that guides the small dsRNAs to its homologous mRNA target. Consequently, RISC cuts the mRNA approximately in the middle of the region paired with the antisense siRNA, after which the mRNA is further degraded. http://www.qbiogene.com/products/transfection/app-sirna.shtml

9. 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 sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair 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 release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.

10. 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.

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

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

16. The function of miRNAs appears to be in gene regulation For 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 are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA the inhibits 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 miRNA triggers 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 translation machinery 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.

17. 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 bloc transcription of homologous promoters (TGS), whereas those corresponding to transcribed sequences direct the silencing machinery to degrade homologous RNAs (PTGS). TGS accompanied by chromatin methylation and remodeling; and PTGS associated with RNA degradation can neutralize endogenous and exogenou invaders such as transgene, viruses and transposon.