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The term gene silencing refers to the transcriptional or posttranscriptional repression of gene expression, which results in a gene that is usually switched on under normal circumstances being turned off by an epigenetic process. Functionally, gene silencing regulates endogenous genes and acts as a defence mechanism, protecting the genome from viruses and transposons. Transcriptional gene silencing occurs due to modifications in histone proteins, the major protein component of chromatin. These modifications form a heterochromatin environment in the vicinity of a gene, causing the gene to be inaccessible to enzymes and proteins involved in transcription. (Redberry 2006:7) Gene silencing at the post transcriptional level occurs due to suppression or excision of the messenger RNA (mRNA) of a certain gene, and commonly occurs by the process of RNA interference (RNAi).RNA interference silences gene expression through the action of small double stranded RNA fragments, approximately 21-25 base pairs in length. Small interfering RNA (siRNA) and microRNA (miRNA) are two different classes of these short, double stranded RNA molecules that are known to be posttranscriptional gene expression regulators (Tang 2005)
In many eukaryotic genomes, endogenous miRNA is encoded (Pollard, Earnshaw 2008:292). These genes are often situated in areas that were formerly considered not to contain any genes. MicroRNA is transcribed as a primary transcript of approximately 60-70 nucleotides in length, which forms a stem-loop structure (Tropp 2008:816). The primary miRNA is then cleaved in the nucleus of the cell, by a bidentate ribonuclease (RNase III) enzyme called Drosha (Bernstein, Caudy, Hammond, Hannon 2001) and "actively transported from the nucleus to the cytoplasm by RNA-GTP and the export receptor Exportin-5" (Tropp 2008:816). Once in the cytoplasm the pre-miRNA undergoes further cleavage by a second double strand specific RNase-III like enzyme Dicer, forming mature miRNAs, which contain a 2nucleotide overhang on the 3' end of each RNA strand (Gabauer, Hentze 2004). In plants, fungi and nematodes, there is a possibility that miRNA may also be synthesised by the enzyme cellular RNA-dependent RNA polymerase; however in flies and mammals there are currently no known homologs of the enzyme that are involved in RNA interference (Bartel 2004).
One strand of the mature miRNA, called the "guide strand" is then incorporated into a multi-protein complex located in the cytosol called the miRNA- induced silencing complex (miRISC), while the other strand is degraded. This complex is the effecter of RNA interference and contains a protein from the argonaute family, which catalyses the hydrolysis of RNA. (Tropp 2008:812). This argonaute protein also selects the strand to integrate into the miRISC, most probably by recognition of the 2nucleotide 3' overhang of the miRNA strand (Peters, Meister 2007), although this is not known for certain. The miRNA guide strand is used as a template, by which the miRISC identifies complementary messenger RNA sequences. Two modes of action of the miRISC effecter complex are understood, with speculation of a third.
It appears the main way miRNA silences gene expression is through repression of mRNA translation. This is supported by the fact that the target mRNA stays intact once miRNA is bound. This process occurs when the homology between the miRNA guide strand and the mRNA strand is incomplete. (Gabauer, Hentze, 2004) The mRNA sequences targeted by the RISC are located in different regions of the mRNA molecules in plants and animals. The sequences (often present multiple times) are located in the 3' untranslated regions (3'UTR) of the mRNA in animals, whereas in plants they are found in either one of two places: the mRNA coding region or within the 5' untranslated region (5'UTR). (Snustad, Simmons 2006:613). The fact that the match between the miRNA guide strand and the target mRNA is not exact often results in several miRNA's binding to the target mRNA to impede translation.
At the present moment in time, it is not fully understood how translation is blocked once the guide miRNA is bound to the 3' UTR of the mRNA. Current experiments suggest that processing bodies (regions located in the cell cytoplasm that contain numerous enzymes that take part in mRNA turnover) play a part in gene silencing that is mediated by miRNA. It is still unknown what role p bodies play in the process, if the "components associated with P bodies execute the silencing step or that the target RNA is directed to P bodies for degradation after miRNA-induced translational repression." (Chendrimada, Finn, Ji, Baillat, Gregory,Liebhaber, Pasquinelli, Shiekhattar 2008:823). Research by Chendrimada et al has also been conducted to see if the protein encoded from the gene eukaryotic translation initiation factor 6 (eIF6), working with miRNA, helps to suppress the translation of the target mRNA. This protein, a translation initiation factor, is known to prevent the formation of the 80S ribosomal subunit from the 40S and 60S subunits, by binding to the 60S subunit. Thus this protein could potentially prevent translation of mRNA by obstructing initiation (Chendrimada et al 2008).
The second way in which miRNA can silence gene expression occurs if the homology between the miRNA guide strand and the target mRNA is complete. In this instance, the guide miRNA directs the catalytically active argonaute protein located in the miRISC to cleave the target mRNA. The action of the argonaute protein hydrolyses the mRNA, destroying it. When the miRNA dictates mRNA cleavage, the mRNA is always sliced in exactly the same place- between the mRNA nucleotides pairing to the tenth and eleventh residues of the miRNA guide strand (Bartel 2004).
The other short double stranded RNA molecule involved in RNAi is called short interfering RNA (siRNA). Unlike miRNA, which is encoded in the genome, siRNA is made by transcribing elements already present in an organism's genome, for example transposons or transgenes (Snustad, Simmons 2006:614). It can also be formed from double stranded RNA, which is made when two transcripts from opposite strands of repeated DNA sequences anneal, or by RNA viruses (Tropp 2008:816). Similarly to miRNA formation, the double stranded RNA is then cleaved by the enzyme Dicer to produce short, mature siRNA fragments, which also contain the 2nucleotide overhang at the 3' end of each RNA strand.
As in the miRNA gene silencing pathway, one strand of the siRNA becomes incorporated into a RISC (this time, it is known as a siRISC), while the other is degraded. The structure of the siRISC is similar to that of the miRISC, and includes an argonaute protein to catalyse RNA hydrolysis. In siRISC the argonaute protein is known to be Ago2 (Dykxhoorn, Palliser, Lieberman 2006). The siRISC functions in a similar manner to the miRISC, using the guide siRNA strand as a template to identify complementary mRNA sequences (Tropp 2008:811). There are two known methods of siRNA gene silencing, one similar to that of miRNA gene silencing, the other different.
The prime way in which siRNA mediates gene silencing occurs when there is perfect, or very near perfect, homology between the siRNA and mRNA target. When this occurs, the guide siRNA strand directs the siRISC complex to the complementary mRNA sequence, and the catalytic Ago2 protein cleaves the mRNA strand at one site. (Tropp 2008:811). The site of cleavage in siRNA gene silencing is the same as in miRNA gene silencing, between the mRNA nucleotides pairing to the tenth and eleventh residues of the siRNA guide strand. Thus, the cleavage of mRNA by either of the short, double stranded RNA molecule appears to be calculated relative to the siRNA or miRNA residues, not as a result of the mRNA base pairing to miRNA or siRNA targets (Bartel 2004).
The silencing of mRNA by siRNA cleavage is very similar to the silencing of mRNA by miRNA, when the homology between the miRNA and mRNA is complete. However, unlike miRNA, siRNA is unable to suppress translation of mRNA when the homology between the siRNA guide strand and the mRNA is incomplete. Therefore if silenced by siRNA fragments, the target mRNA is always destroyed, yet if silenced by miRNA, providing incomplete homology, the mRNA remains intact.
Whereas all gene silencing by miRNA occurs in the cytoplasm, silencing by siRNA can occur in either the cytoplasm or the nucleus of a cell. RNA is able to identify and form duplexes with RNA and DNA, and thus RNAi can exert its effects on genomic DNA in the nucleus (Berezhna, Supekova, Supek, Schultz, Deinz 2006).
Scientific experiments have shown that siRNA can play a role in DNA methylation, resulting in transcriptional gene silencing. This is known as RNA-directed DNA methylation (RdDM), and results in de novo methylation of the DNA sequence, and is particularly prominent in plants (Pelissier, Wassenegger 2000). Research conducted by Kawasaki and Taira in 2004 shows that this can also occur in human cells, as well as in plants and many animals. In mammalian cells methylation typically occurs on cytosine residues found in regions of cytosinse-phosphate-guanine sequences (CpG) sites. However in plant cells DNA methylation can also occur at two other sites, CpNpG and CpNpN, where N is any of the nucleotide bases except guanine. In both plants and animals, the addition of a methyl group to one of the nitrogenous bases of DNA results in the prevention of DNA transcription. This could be because the access of the enzyme RNA polymerase to the DNA is decreased (Robinson 2006), although the mechanisms of siRNA DNA methylation are still not fully understood.
At the present moment in time, the fact siRNA can induce DNA methylation to silence genes and miRNA cannot is a difference in their methods of gene silencing. However, current research, primarily conducted on plants, puts forth the idea that miRNA may also be able to cause DNA methylation, resulting in gene silencing. A news article written by Ronemus and Martienssen (2005) summarizes the main developments in understanding the process of miRNA DNA methylation. Studies conducted on Arabidopsis plants (by the group of scientists Bao, Lye, Barton 2004) show that methylation occurs in genes which normally have their expression diminished in the presence of miRNAs. When mutations were present in the miRNA binding sites of these genes, the levels of methylation decreased and a "de-silencing" effect was observed (Ronemus, Martienssen 2005).Thus Bao, Lye and Barton (2004) put forth the conclusion DNA methylation in these genes relied on miRNA molecules and suggested possible mechanisms of miRNA induced DNA methylation, ultimately resulting in gene silencing (Ronemus, Martienssen 2005). The mechanisms they suggested was that the miRNA in the cell cytoplasm re-enters the cell's nucleus with a miRISC, which then binds to a complementary mRNA strand generated by the genes in question. Using the complementary mRNA, the miRISC may "recruit 'chromatin-remodelling' machinery to the DNA to achieve methylation." (Ronemus, Martienssen 2005: Figure 1a legend). An alternative theory also proposed by Bao et al and cited by Ronemus and Martienssen (2005) is that mRNA is directed to the DNA to provoke methylation after it has encountered miRNA cleavage. However, the exact method as to how miRNA induces DNA methylation, promoting gene silencing is still unknown, but there is a strong possibility that it may exist, like siRNA induced DNA methylation gene silencing does.
There are many similarities and differences in miRNA mediated and siRNA mediated gene silencing. MicroRNA and siRNA differ in how they are synthesized, the amount of homology required between themselves and their target mRNA and the main way they execute their silencing effects. They also differ in the types of silencing they specify. MicroRNA is formed from genes which denote the repression of vastly different genes; this is known as "hetero-silencing." On the other hand, "auto-silencing" normally occurs with siRNA, silencing the identical, or very similar, locus which they initially originate from (Bartel, 2004). MicroRNA has the ability to silence genes when the homology between itself and the mRNA is incomplete, whereas siRNA can only exert its affects when there is complete homology between itself and its mRNA target. MircoRNA predominately silences genes by repressing the translation of mRNA by binding to it, which occurs with incomplete homology between the miRNA guide and the mRNA target. On the contrary, siRNA mainly represses gene expression by cleaving the mRNA target between the nucleotides that base pair to the tenth and eleventh siRNA residues. This results in the mRNA being degraded, and can only occur with complete homology between the two sequences. This cleavage of the mRNA target can also occur in miRNA mediated gene silencing, and occurs, like siRNA mediated repression, when there is complete homology between the miRNA template and the mRNA target. Small Interfering RNA also has the ability to repress gene expression at the genome sequence level, by forming an RNA DNA duplex. Methylation of the DNA occurs, resulting in gene expression being inhibited. There is speculation that miRNA may also be able to initiate DNA methylation, and hence cause gene repression, however this is yet to be determined for certain.