Gene silencing is a general term used in reference to suppression of gene expression. Applicable to both transcriptional and translational levels, this mechanism occurs without resulting to genetic modification. The cellular machinery employs a “switch off” mechanism that blocks active gene product formation [1]. An example of this gene regulation that endogenously occurs is called RNA interference (RNAi). This is a process utilized by biological systems at the post transcriptional level.
When the body is invaded by foreign organisms, the internal environment becomes exposed to a different genetic material. Upon detection, the body elicits a specific immune response in order to silence the effect of the invading species’ nucleic acid sequences. Being a usual response against viral and transposon attacks, this innate mechanism prevents the occurrence of negative impacts of cellular activity interruption in the host genome [1]. This phenomenon is currently found to be manifested in all eukaryotic organisms.
Observed from the simplest forms of eukaryotes, such as yeast, up to the most complex, such as mammals, double stranded RNA (dsRNA) causes messenger RNA (mRNA) degradation in a sequence-specific manner. It is actually a natural antiviral response of cells that can be used to inhibit the function of specific target genes that frequently include cancer, AIDS, and hepatitits [2]. The ability of RNAi to interrupt and suppress genetic expression has paved for its utilization as a tool for reverse genetics in eukaryotes.
It proves to be a significant research tool that also provides a better characterization of already studied and known genes. It is a potent instrument in the conception of a more thorough and a deeper understanding of biological function through the synthesis of accumulated genomic information. It has revolutionized biological research and has led to prospective development of treatment and drugs to presently incurable diseases. The ability of RNA to selectively silence genes is considered a scientific breakthrough that has served as a precursor to further genetic discoveries [2].
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RNA Interference: History and Discovery Studies on RNA interference primiarily started when a research was conducted to genetically modify the coloration of Petunias. In the late 1980s, Napoli et al. [18]observed that intensifying the violet hue of the flowers instead resulted to the expression of a white phenotype. The white and variegated plants may somehow have been the consequence of a decreased expression, a opposed to the anticipated increase, upon injection of extra copies of the genes [3]. The introduction of a powerful promoter prevented the expression of both the introduced and the homologous endogenous gene.
This said phenomenon is otherwise known as cosuppression . This term emerged in order to refer to the the ability of exogenous elements to alter the expression of endogenous genes [15] cosuppression is subsequently observed in various species of plants and fungi (where it is called quelling ) occurring at the post transcriptional level [12]. This gene silencing mechanism was identified to have occurred at the post-transcriptional level. This post-transcriptional gene silencing (PTGS) is mediated by a diffusible and trans-acting molecule in Neurospora and plants.
In 1998, Fire et al. Discovered that dsRNA is a potent inducer of RNA interference in their studies conducted on Caenorhabditis elegans and Neurospora. This is a pivotal study as it revealed approaches on efficient gene silencing induction in C. elegans and other organisms. This expedited the development of a unifying mechanism that underlies a host of cellular pathways. However, this proves deficient in providing substantial explanation on the triggering mechanism by dsRNA in order for the science community to officially accept the idea [19].
Before, scientist thought that dsRNA is a nonspecific silencing agent that codes for mRNA destruction which therefore results to the suppression of protein translation in cells of mammalian species. These double stranded RNAs were also thought to possess stable energy levels and are incapable of base pairing. With this, a model was constructed that illustrates the activation of sequence-specific silencing using dsRNA, implying the existence of cellular mechanisms for unwinding the dsRNA and promoting the search for complementary base-pairing partners among the vast collection of nucleic acid sequences available.
The rapid acceptance of the proposal that this dsRNA is responsible for RNA interference can be attributed to the research conducted by Guod and Kemphues. They attempted to use the antisense-mediated silencing technique where they blocked par-1 expression of C. elegans par-1 mRNA. This caused the delivery of large amounts of nucleic acids with complementary sequences to the target messenger RNA into the cellular cytoplasm.
This base pairing between the sense and antisense sequences was originally thought to cause the passive blockage of mRNA translation, or it also can possibly result in the recruitment of nucleases that promote mRNA destructions. Instead, both the control sense and the antisense caused the induction of gene silencing [15]. In a study conducted by Fire and Mello, they tested the RNA effect on the phenotype of the nematode C. elegans. Their results revealed that annealed sense and antisense RNA expressed the desired phenotype, but neither sense nor antisense alone produced the expected results (Figure 2).
Their findings also proved that the injection of dsRNA would lead to the degradation of target mRNA, which is an efficient way to trigger gene silencing. Another conclusion in their research is that there is a specific mRNA homologous to the dsRNA in gene silencing and that mRNA are unaffected. When the dsRNA was injected into the subject animal, it has to correspond to the mature mRNA sequence cause neither the intron nor the promoter sequences will trigger a response. This leads to the probability that this is a cytoplasmic mechanism occurring at the posttranscriptional level.
Another conclusion to their experimental results was that when the mRNA disappeared, it was presumed to have been destroyed. They found in their study that a few dsRNA molecules are sufficient in order to cause full gene silencing. This is an indication that amplification of dsRNA is occurring and that catalytic rather than stoichiometric action of dsRNA happens. They have also observed that the phenotypic effect of the dsRNA could be passed on to the succeeding generations, suggesting that this effect can be transmitted from the parent to the offspring, demonstrating a complete penetrance in the germline.
They concluded their research study with the speculation that dsRNA is a possible physiological gene silencer that organisms can use [15]. A year after this breakthrough discovery, numerous studies have been conducted on other organisms including trypanosomes, plants, and planaria. It was found that RNA interference is generally a phenomenon that is utilized by eukaryotes, with the exception demonstrated by the yeast Saccharomyces cerevisiae [4]. Figure 1. Cosuppression of sense and antisense genes [15] RNA Interference: Mechanism
When the controversial RNA interference mechanism was discovered, a correlation between PTGS and the presence of small RNAs, of approximately 25 nucleotides long, was immediately noticed. The RNAi mechanism was established based on the studies conducted on test subjects such as C. elegans and Drosophila. In organisms other than mammals, dsRNAs of approximately 200 nt long homologous to the target gene can cause RNAi to take into effect. In mammalian systems on the other hand, short synthetic siRNAs are instead utilized as dsRNAs with length longer than 30 nt that induce an antiviral interferon response.
As an alternative procedure, the DNA constructs, coding for short hairpin RNAs (shRNAs) can also trigger RNAi. In current studies, siRNAs and shRNAs are carefully designed to make an efficient gene silencing system with minimal off-target effects [4]. It has been found that dsRNA (Fig. 3) can artificially trigger RNAi pathway. This gene silencing method is considered an ancient antiviral mechanism, passed on from ancestral species to their descendants throughout the evolutionary process.
Since most of lower forms of organisms lack the genetic material DNA and only have RNA, all multicellular organisms do have conserved protein machinery that can be translated from double stranded RNA. Viruses only have RNA and undergo at least a stage in the life cycle that they are able to synthesize double stranded RNA. An enzyme that causes the degradation of this double stranded RNA is called the Dicer. This enzyme, when injected into non-mammalian systems, it breaks long dsRNAs into small RNA segments in the cell’s cytoplasm.
This enzyme that belongs to the RNase III family are about 200 kDa multidomain proteins. Scientists have formulated their probable structure that includes the ATPase/RNA helicase domain, C-terminal dsRNA binding domain, two catalytic RNase III domains, and conserved PAZ domain shared with Argonaute [5]. The end of the dsRNA is speculated to be recognized and to bind with the Dicer’s PAZ domain. The dicer acknowledges the 3’ overhangs and continuously causes a 21-25 bp intervals of dsRNA cleavage. Consequently, the product is a dinucleotide dsRNA with 3’ overhangs and phosphorylated 5’ ends.
When shRNAs are used to induce gene silencing, the process of shRNA entry into the RNAi pathway is apparently affected by the length of the hairpin. The dicer are said to cleave 29 bp stems into siRNA pieces of 21-22 nt in length, however, shRNAs with 19 bp stems are not completely broken into segments. This indicates that siRNAs and shRNAs are of exogenous origin with short stems only instructed for entry into the RNAi pathway after the initial processing of dicer [5]. Figure 2. RNA Interference Pathway [19] The process continues through the usage of RISC enzyme complex.
RISC stands for RNA Induced Silencing Complex that is used by cells in order to utilize the RNA segments produced by the dicer. These short pieces of RNA would serve as a template to find and destroy single stranded RNA with the same sequence. Viruses that copy mRNA sequences to direct their protein synthesis exemplify this procedure. Both the dicer and RISC compose the system of RNA interference. In short, both enzymes are used when dsRNA is recognized and becomes a pattern to silence gene expression of similar sequences through mRNA transcript destruction occurring at the posttranscriptional level [6].
The dicer, Dcr-2, and the small dsRNA binding partner, R2D2, collaborate for the direction of siRNA to RISC components. These two are bound by a heterodimer that they themselves synthesized. Here, the more stable end of siRNA is bound to the thermodynamically unstable end of Dcr-2. This asymmetry of thermodynamic stability determines the strand to be loaded into RISC [7]. Because of the phosphate present in the 5’ end of an siRNA, there is an increased probability of it binding with R2D2.
The heterodimer generated by the Dcr-2 and R2D2 participates in a RISC loading complex (RLC), which is an intermediate where Argonaute protein Ago2 gradually displaces Dcr-2/R2D2 complex [6]. In the effector or silencing step of the RNAi pathway, RISC components assemble with guide strands to make a mature RISC. This mature RISC (siRISC) functions as a multiple turnover enzyme[7]. Upon breakdown of targeted mRNA, siRISC is dissociated to continue the cleavage cycle. This is a spontaneous process that does not require energy expenditure but it speeds enzyme turnover. The main catalyst for mRNA cleavage is found to be Argonaute (Fig.
4) [10]. Figure 5. Assembly and mRNA Cleavage Activity [10] Besides functioning as an antiviral system, RNAi has a significant role in maintaining genomic order through the suppression of movement of genetic elements that are capable of translocating. Examples of these mobile elements are transposons and repetitive sequences. Now, the possibility that RNAi can regulate and improve normal cellular gene expression is being recognized [10]. Most of the research conducted regarding RNAi were done on invertebrate species. When this mechanism was tested on human subjects, the results did not meet the expected and desired output.
However, a pivotal discovery emerged when short dsRNA molecules of 30 nt in length were observed to fail the induction of interferon response. Since gene expression was not shutdown by the msall interfering RNAs, they instead were observed to have the ability of direct a sequence specific degradation of homologous mRNA following a similar procedure compared to plants, worms and insects. When introduced into mammalian cellular systems, 20 nt long siRNAs directly cause the RISC to initiate gene silencing with the same sequence. Even after the interferon system has evolved, its parsimonious nature has reserved RNAi as a back-up system [10].