RNAi & Intronic microRNA Biogenesis
Overview of RNA Interference
RNA interference (RNAi) is a post-transcriptional gene silencing (PTGS) process in which small RNA fragments mediate the degradation of target messenger RNAs (mRNA) and prevents protein translation as a result. RNAi gained worldwide recognition in 1998 when Fire et al. demonstrated this phenomenon by introducing dsRNAs into C. elegans and showed that this resulted in potent gene silencing. In contrast to antisense-mediated gene silencing, RNAi is dependent on a catalytic mechanism to achieve effective gene silencing. Protein translation inhibition is not achieved strictly by physical binding of the antisense strand (single-stranded RNA fragments) to mRNA, but rather in a post transcriptional process. Smaller RNA strands, or fragments, in the nucleus associate with a number of mRNA-degrading proteins to form a RNA-induced silencing complex (RISC) that in turn recognize and bind to respective target mRNA strands. The mRNA is subsequently degraded and protein translation is halted as a result.
The small RNA fragments that are responsible for mRNA targeting specificity consist of short interfering RNAs (siRNA) and microRNAs (miRNA). siRNAs are typically 20-25 nucleotides long and double stranded RNAs (dsRNA), whereas miRNAs are 21-23 nucleotides long and single-stranded RNA molecules. In its initial incarnation, RNAi lacked a side-effect that resulted from anti-sense annealing. In traditional antisense annealing, there is a chance of phenotype mutation, due to the “knock-out” of a targeted gene . During RNAi, however, the gene is selectively downregulated in such manner that minimal phenotype mutation occurs. This typically leads to a much lower mortality rate of the host cell. siRNAs were first discovered in 2001 by Jones et al. and were labeled as part of PTGS in plants . Subsequently, their finding was translated into mammalian cells to show RNAi characteristics . Given the enormous implications of these findings, a greater attention is now placed on this method of gene regulation. The innovation surrounding RNAi is that it’s highly specific, potent, and can traverse to other cells far from site of introduction.
By definition, siRNAs (small-interference RNA) are commonly expressed as the exogenous, synthetic small RNA whereas miRNAs is the endogenous genome-encoded small RNA. Despite the fact that both induce RNAi effects, the two differ in several ways. siRNAs, as mentioned before, are exogenous molecules that are introduced into the host cells to induce a series of responses. The siRNA strands are also perfectly complementary to the targeted mRNA whereas miRNAs is only partially complementary. With regard to regulation, this perfect complementary leads to a complete shut-off, or silencing of a gene, while partial complementary leads to a repression of a gene. Similarly, both siRNAs and miRNAs bind with the multi-protein RISC-like and Argo-like proteins that cleave the target mRNA. The attractiveness of siRNAs is that they offer researchers greater flexibility and creativity, since they can be produced synthetically and fine-tuned for more precise targeting. But unfortunately, like a double edged sword, the attractiveness of siRNA is also its weakness [5-13]. Coincidently, evolutionary widespread miRNAs exhibit the same RNAi attributes and as siRNAs . Despite its perfect complementary and assumed high specificity, tendency of off-target effects and cell cytotoxity has been shown in studies.  In a 2004 study of siRNA off-targeting, performed a large scale microarray profile that concluded with 1.5 to 3 fold changes in gene expression after siRNA transfection.  Initial reception of these findings by the scientific community felt these occurrences were modest at best and insignificant, but studies that followed proved otherwise. This suggests that siRNAs are more suitable for research purposes but may not yet be suitable for clinical/therapeutic applications unless a solution is found to address their off-target effects.
Thus far, siRNAs have only been identified in lower mammalians and plants, and this presents another limiting factor to its potential for therapeutic applications. Results from a number of studies have also revealed the cytoxicity of siRNAs in human cells due to their double-stranded nature , which has been shown to activate the interferon-induced non-specific RNA degradation and subsequent protein kinase PKR and 2-5A pathways that trigger cell death via apoptosis . In another study of RNA splicing-mediated gene silencing has revealed another contributing factor of mammalian cells’ sensitivity to siRNA, the transcriptional polymerase. Specifically, several vector-based RNAi expression systems rely on type-III RNA polymerase for their transcriptional duties . Despite the strategy to use Pol III, its ubiquitous nature also leads to a lack of cell specificity . Perhaps the incorporation of a novel RNA polymerase along with modified miRNA would promote more stability? Researchers have derived such an expression system that serves as a powerful tool for targeting and silencing specific genes. By utilizing type-II RNA polymerase in place of type-III, intron-derived miRNA has exhibited success in shutting off genes .
The process of DNA transcription synthesizes a premature mRNA (pre-mRNA) consisting of introns and exons. Subsequent RNA splicing removes the intron portion from the pre-mRNA and the exons are ligated together to form the mature mRNA for protein translation. The notion that introns are genetic wastes with no definted functions was recently corrected by the discovery of intron-derived, or intronic, miRNA. Lin et al. were the first to identify intronic miRNAs and to successfully demonstrate their ability to induce RNAi in many cell types in vivo. It is important to note that miRNA is literally a re-insertment of what is naturally found in the cell, but slightly modified. These modified miRNA’s need to be in primary transcript form (pri-mRNA) such that it can be excised to become a shorter chain (pre-miRNA) with the desired miRNA segments still intact. In the nucleus, Pri-miRNA is transcribed from DNA by RNA polymerase II or III, depending on the expression system, to form pri-miRNA, which is then followed by excise by RNase III endonuclease Drosha . The RNase III Drosha then cleaves the pri-miRNA into pre-miRNA that is then transported to the cytoplasm by RAN-GTP and Exportin-5 . A Dicer-like endonuclease then splices the pre-miRNA into mature miRNAs (21-23 nucleotide long). These small mature miRNA fragments are then incorporated into RNP (Ribonulcear particle) where the two form the RISC complex. When the Dicer-like endonuclease cleaves the pre-miRNA, only one of the two complementary RNA strands is integrated with the RISC complex . The responsibility of selecting the guiding strand to incorporate into the RISC belongs to the Argonaute protein, which is the RNase in the RISC complex. The passenger, or the antisense strand, is subsequently degraded. The RISC complex identifies and binds with target mRNA molecule and execute its degradation. Although the processes involved in intronic miRNA- and siRNA-mediated RNAi are similar, it is important to keep in mind that intronic miRNAs are strictly dependent on Pol-II-mediated DNA transcription and RNA splicing for their biogenesis.
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