Gene silencing is a general term used to describe the epigenetic regulation of gene expression. In particular, this term refers to the ability of a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research.  In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and diseases, such as infectious diseases and neurodegenerative disorders.
Gene silencing is often confused with gene knockout. Though gene silencing is the same as gene knockdown, it is different from gene knockout.  When genes are knocked down, their expression is reduced.  In contrast, when genes are knocked out, they are completely erased from the organism's genome and, thus, have no expression.  Gene silencing is considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, generally reduce the expression of a gene by at least 70% but do not completely eliminate it. Methods using gene knockdowns are often considered better than gene knockouts since they allow researchers to study essential genes that are required for the animal models to survive and cannot be removed. In addition, they provide a more complete view on the development of diseases since diseases are generally associated with genes that have a reduced expression.
- 1 Gene silencing in cells
- 2 Gene silencing methods used in research
- 3 Gene silencing in medical research
- 4 External links
- 5 References
Gene silencing in cells
Transcriptional gene silencing
- Genomic Imprinting
- Transposon silencing
- Transgene silencing
- Transcriptional gene silencing
- position effect
- RNA-directed DNA methylation
Post-transcriptional gene silencing
Meiotic gene silencing
Gene silencing methods used in research
Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson. Oligonucleotides, which are short nucleic acid fragments, bind to complementary target mRNA molecules when added to the cell. These molecules can be composed of single-stranded DNA or RNA and are generally 13-25 nucleotides long. The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism. RNase H-dependent oligonucleotides cause the target mRNA molecules to be degraded, while steric-blocker oligonucleotides prevent translation of the mRNA molecule. The majority of antisense drugs function through the RNase H-dependent mechanism, in which RNase H hydrolyzes the RNA strand of the DNA/RNA heteroduplex. This mechanism is thought to more efficient, resulting in an approximately 80% to 95% decrease in the protein and mRNA expression.
Ribozymes are catalytic RNA molecules used to inhibit gene expression. These molecules work by cleaving mRNA molecules, essentially silencing the genes that produced them. Sidney Altman and Thomas Cech first discovered catalytic RNA molecules, RNase P and group II intron ribozymes, in 1989 and won the Nobel Prize for their discovery. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs are generally found in viruses or viroid RNAs. These motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule. Lower eukaryotes and a few bacteria contain group I and group II ribozymes. These motifs can self-splice by cleaving and joining together phosphodiester bonds. The last ribozyme motif, the RNase P ribozyme, is found in Escherichia coli and is known for its ability to cleave the phosphodiester bonds of several tRNA precursors when joined to a protein cofactor.
The general catalytic mechanism used by ribozymes is similar to the mechanism used by protein ribonucleases. These catalytic RNA molecules bind to a specific site and attack the neighboring phosphate in the RNA backbone with their 2’ oxygen, which acts as a nucleophile, resulting in the formation of cleaved products with a 2’3’-cyclic phosphate and a 5’ hydroxyl terminal end. This catalytic mechanism has been increasingly used by scientists to perform sequence-specific cleavage of target mRNA molecules. In addition, attempts are being made to use ribozymes to produce gene silencing therapeutics, which would silence genes that are responsible for causing diseases.
RNA interference (RNAi) is a natural process used by cells to regulate gene expression. It was discovered in 1998 by Andrew Fire and Craig Mello, who won the Nobel Prize for their discovery in 2006. The process to silence genes first begins with the entrance of a double-stranded RNA (dsRNA) molecule into the cell, which triggers the RNAi pathway. The double-stranded molecule is then cut into small double-stranded fragments by an enzyme called Dicer. These small fragments, which include small interfering RNAs (siRNA) and microRNA (miRNA), are approximately 21-23 nucleotides in length.  The fragments integrate into a multi-subunit protein called the RNAi induced silencing complex (RISC), which contains Argonaute proteins that are essential components of the RNAi pathway.  One strand of the molecule, called the "guide" strand, binds to RISC, while the other strand, known as the "passenger" strand is degraded.  The guide or antisense strand of the fragment that remains bound to RISC directs the sequence-specific silencing of the target mRNA molecule. The genes can be silenced by siRNA molecules that cause the endonucleatic cleavage of the target mRNA molecules or by miRNA molecules that suppress translation of the mRNA molecule. With the cleavage or translational repression of the mRNA molecules, the genes that form them are essentially inactive. RNAi is thought to have evolved as a cellular defense mechanism against invaders, such as RNA viruses, or to combat the proliferation of transposons within a cell’s DNA. Both RNA viruses and transposons can exist as double-stranded RNA and lead to the activation of RNAi. Currently, siRNAs are being widely used to suppress specific gene expression and to assess the function of genes.
Gene silencing in medical research
RNA interference has been used to silence genes associated with several cancers. In in vitro studies of chronic myelogenous leukemia (CML), siRNA was used to cleave the fusion protein, BCR-ABL, which prevents the drug Gleevec (imatinib) from binding to the cancer cells. Cleaving the fusion protein reduced the amount of transformed hematopoietic cells that spread throughout the body by increasing the sensitivity of the cells to the drug. RNA interference can also be used to target specific mutants. For instance, siRNAs were able to bind specifically to tumor suppressor p53 molecules containing a single point mutation and destroy it, while leaving the wild-type suppressor intact.
Receptors involved in mitogenic pathways that lead to the increased production of cancer cells have also been targeted by siRNA molecules. The chemokine receptor chemokine receptor 4 (CXCR4), associated with the proliferation of breast cancer, was cleaved by siRNA molecules that reduced the number of divisions commonly observed by the cancer cells. Researchers have also used siRNAs to selectively regulate the expression of cancer-related genes. Antiapoptotic proteins, such as clusterin and survivin, are often expressed in cancer cells. Clusterin and survivin-targeting siRNAs were used to reduce the number of antiapoptotic proteins and, thus, increase the sensitivity of the cancer cells to chemotherapy treatments. In vivo studies are also being increasingly utilized to study the potential use of siRNA molecules in cancer therapeutics. For instance, mice implanted with colon adenocarcinoma cells were found to survive longer when the cells were pretreated with siRNAs that targeted B-catenin in the cancer cells.
Viral genes and host genes that are required for viruses to replicate or enter the cell, or that play an important role in the life cycle of the virus are often targeted by antiviral therapies. RNAi has been used to target genes in several viral diseases, such as the human immunodeficiency virus (HIV) and hepatitis.  In particular, siRNA was used to silence the primary HIV receptor chemokine receptor 5 (CCR5). This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells.  A similar technique was used to decrease the amount of the detectable virus in hepatitis B and C infected cells. In hepatitis B, siRNA silencing was used to target the surface antigen on the hepatitis B virus and led to a decrease in the number of viral components. In addition, siRNA techniques used in hepatitis C were able to lower the amount of the virus in the cell by 98%. 
Gene silencing techniques have also been used to target other viruses, such as the human papilloma virus, the West Nile Virus, and the Tulane virus. The E6 gene in tumor samples retrieved from patients with the human papilloma virus was targeted and found to cause apoptosis in the infected cells. Plasmid siRNA expression vectors used to target the West Nile Virus were also able to prevent the replication of viruses in cell lines. In addition, siRNA has been found to be successful in preventing the replication of the Tulane virus, part of the Caliciviridae family of viruses, by targeting both its structural and non-structural genes. By targeting the NTPase gene, one dose of siRNA 4 hours pre-infection was shown to control Tulane virus replication for 48 hours post-infection, reducing the viral titer by up to 2.6 logarithms. Although the Tulane virus is species-specific and does not affect humans, it has been shown to be closely related to the human norovirus, which is the most common cause of acute gastroenteritis and food-borne disease outbreaks in the United States. Human noroviruses are notorious for being difficult to study in the laboratory, but the Tulane virus offers a model through which to study this family of viruses for the clinical goal of developing therapies that can be used to treat illnesses caused by human norovirus.
Unlike viruses, bacteria are not as susceptible to silencing by siRNA. This is largely due to how bacteria replicate. Bacteria replicate outside of the host cell and do not contain the necessary machinery for RNAi to function. However, bacterial infections can still be suppressed by siRNA by targeting the host genes that are involved in the immune response caused by the infection or by targeting the host genes involved in mediating the entry of bacteria into cells. For instance, siRNA was used to reduce the amount of pro-inflammatory cytokines expressed in the cells of mice treated with lipopolysaccharide (LPS). The reduced expression of the inflammatory cytokine, tumor necrosis factor α (TNFα), in turn, caused a reduction in the septic shock felt by the LPS-treated mice. In addition, siRNA was used to prevent the bacteria, Psueomonas aeruginosa, from invading murine lung epithelial cells by knocking down the caveolin-2 (CAV2) gene. Thus, though bacteria cannot be directly targeted by siRNA mechanisms, they can still be affected by siRNA when the components involved in the bacterial infection are targeted.
- RNAiAtlas - database of siRNA libraries and their target analysis results
- Science project: Transgenic apple varieties Approaches to preventing outcrossing – possible effects on micro-organisms
- Gene silencing at the US National Library of Medicine Medical Subject Headings (MeSH)
- Research project: New Cost-effective method for gene silencing
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