Silencers
RNA silencing is an evolutionary conserved intracellular surveillance system based on the recognition and targeting of RNAs containing regions that are double stranded. Typical roles of RNA silencing include genome defense and specification of heterochromatin formation, posttranscriptional inhibition of gene expression by miRNA and antiviral defense. RNA silencing is induced by double-stranded RNA that is sensed by the enzyme Dicer. Dicer digests the double strand into double-stranded strand silencing interfering silencer RNA which incorporates with RNA-induced silencing complex (RISC) (Ahlquist, p. 1270). There are two classes of silencers both of which diminish a gene’s expression level. The first class is composed of position-independent elements that direct an active repression mechanism, although the functional basis for the repression by most members of this class remains uncharacterized. The second class is composed of negative regulatory elements (NREs) that are position-dependent sequences with a passive repression mechanism that seems to be demarcated by changes in histone acetylation. Repression is thought to result from physical inhibition of the interaction of transcription factors with other DNA sequence motifs by specific silencer-bound proteins. Silencer elements have been identified in a variety of positions in human genes, both near the promoter and within introns, exons, or flanking sequences. In some mammalian systems, the silencers located within the exon function at the level of DNA for regulation of gene transcription and also at the level of RNA for control of translation through interference with normal RNA processing or trafficking (Ahlquist, p. 1273).
Boundary Elements
Boundary elements also referred to as insulator elements are regions of DNA from 0.5 to 0.3 kb long that demarcate the 5 inches and 3 inches margins of a gene. Boundary elements function by establishing an intricate three-dimensional chromatin structure that is important for DNA and RNA processing. Boundary elements also block the inappropriate repression of transcription caused by local silencers.
RNA interference (RNAi)
RNA interference (RNAi) is the ability to selectively silence the genes and has revolutionized microbiology. Recent studies have shown that a cell that is a double-stranded RNA copy of a specific gene will prevent the native copy of that gene from being expressed. It is now possible for researchers to carry out studies on the function of any gene by silencing it with RNAi followed by monitoring how the cell’s functions are impacted. RNAi has first discovered accidentally in petunia plants in 1990 in Oakland, California at the DNA Plant Technology Corporation. RNAi is one of the tools of the cell to regulate gene expression and it occurs naturally. In 1998 two scientists, Andrew Fire and Craig Mello characterized the mechanism of gene silencing. This particular experiment was carried out on worms and it demonstrated that the double-stranded RNA is the key player. This meticulous work led to the pair winning the 2006 Nobel Prize in Physiology or medicine. According to Fire, RNAi plays an important role in fighting viral infections and in the molecular changes that cause cells to become cancerous. The RNAi mechanism typically involves recognition and response. A cell on seeing the double-stranded RNA will first respond by chopping it into bits which is recognizable since the double-stranded RNA is a distinguished structure when viruses replicate. However, the cell may go a step ahead and chop any stuff that looks like the RNA even those that are single being-stranded. A variety of short double-stranded or single-stranded RNA molecules has recently been shown to have a role in the regulation of gene expression. RNA-mediated or interference control of gene expression constitutes an entirely new pathway of gene regulation and occurs through several different mechanisms. The RNA-interference (RNAi) has been suggested as a novel technique for treating a wide variety of illnesses ranging from cancer to infectious diseases. Several model systems have been developed and they demonstrate the potential of RNAi-based approaches despite the fact that delivery of short RNA molecules remains an obstacle to RNA-based therapies. For instance, injection of short interfering RNA (siRNA) has been used to protect mice from viral hepatitis and nasally administered siRNA has been used to protect mice from respiratory virus infection (Elbashir, Harborth, Lendeckel, et al., p. 494).
Short interfering RNA
RNAi through short interfering RNA (siRNA) is suggested to have initially developed as a mechanism to silence the expression of genes encoded by repetitive sequence elements. The siRNAs are double-stranded RNA molecules that are 21 to 22 nucleotides long. They are produced from long double-stranded RNA molecules that are cut into shorter fragments by an enzyme named Dicer. An RNA-induced silencing complex (RISC) removes the sense strand leaving the anti-sense to guide the RISC to mRNAs produced from the target gene. The RISC prevents the production of the encoded protein which effectively silences the gene from which the mRNA is produced. The siRNAs are also capable of directly inhibiting gene expression by transcriptional silencing. However, the process of this mechanism remains unclear though it has been suggested that transcriptional silencing involves DNA methylation as well as chromatin remodeling through histone methylation (Ahlquist, 1272).
Micro RNA
Micro RNAs (miRNAs) are short single-stranded molecules that are 19 to 25 nucleotides long. They play vital roles in gene regulation, development and are also evolutionary conserved. In humans, there are approximately 200 to 255 genes encoding miRNAs. The miRNAs inhibit translation of mRNA binding to partially complementary sequences in the 3’ untranslated region of mRNA. Generally, mRNA contains many miRNA binding sites in its 3’ untranslated region, and since binding is by only partially complementary sequences, several Miura can bind to each transcript and each MRA can bind to mRNA transcripts from several different genes. A wide variety of novel classes of short RNAs have recently been discussed such as the small modulatory RNA (mRNA). The mRNAs are short double-stranded RNA molecules that are 20 to 23 nucleotides long and like miRNAs are evolutionarily conserved. Despite the fact that the details of this mechanism are unclear, mRNAs exert control over gene expression via interaction with specific regulatory proteins.
Silencer RNA (siRNA)
The siRNA refers to short or small interfering RNA and requires transfection of cells using lipid-based transfection reagent. In addition, it is also useful for transient reduction. The siRNA gene silencers are groups of three specific 19-25 nucleotide-long double-stranded RNA molecules with 2’ to 3’ hanging on each head. Further, there are 10 µM and about 50-100 transfections. Upon request, single siRNA duplex components are available for independent verification of target gene silencing. The Santa Cruz Biotechnology company gives gene silencers targeted to 23,775 human genes. This is suggested to present approximately 100% of putative protein-encoding human genes. Other offers include 26,654 putative protein-encoding mouse genes which represent 100% (Elbashir, Harborth, Lendeckel, et al., p. 497).
How do siRNA Gene Silencers work
The siRNA gene silencers enter the cell via lipid-based transfection. The siRNA unwinds after binding with the RISC molecules and the double strands are separated. The activated RISC/siRNA complex associates with the target mRNA and gets cleaved into short strands. To counteract with the silencer RNA, viruses develop silencing suppressors that hold up the siRNA-guided RNA silencing pathway. A recent study using heterologous Drosophila in vitro embryo RNA was used to analyze the molecular mechanism of suppression of silencing suppressors. The result showed that different silencing suppressors inhibit the RNA silencing via binding to siRNAs. The efficiency of silencer RNA in humans, mouse, and rat has been tested. However, the success of the RNAi experiment depends only on the transfection. It is therefore used for developing and optimizing the siRNA transfection conditions such as the cell number, concentrations of siRNA and the amount of transfection reagent. There are several support products for siRNA gene silencers include the siRNA transfection reagent, transfection medium, dilution buffer, control siRNA and fluorescein conjugated control siRNAs which presents twisted sequences will not lead to specific knock-down of any known cellular mRNAs (Elbashir, Harborth, Lendeckel, et al., p. 497).
References
- Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. “ “Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.” Nature 411 (2001): 494–498.
- Ahlquist, P. “RNA-dependent RNA polymerases, viruses and RNA silencing.” Science 296 (2002): 1270–1273.