Messenger RNA Interference Pathway

RNA interference, or RNAi, is a biological process in which RNA molecules reduce the gene expression of an organism. This is done typically by causing the destruction of specific mRNA molecules. RNAs are direct products of genes, these small RNAs can bind to other mRNA molecules to either increase or decrease their activity like in the example of preventing an mRNA from producing a protein. There are two types of RNA molecules that are central to RNAi, these molecules are, micro RNA (miRNA) and small interfering RNA (siRNA).

In 1956, Francis Crick first described what he called “The central dogma of molecular biology.” This essentially describes the flow of genetic information within cells. It states that DNA is transcribed into RNA with the help of an RNA polymerase enzyme. The RNA is then translated into a protein by protein synthesis. One thing that could drastically alter the genetic information within cells is a process called gene silencing. This process regulates the gene expression of certain genes and can occur in either transcription or translation. The process has been coined RNA interference and dsRNA gene silencing (Davidson and McCray Jr. 2011). Since direct evidence of double stranded RNA’s role in gene silencing was found in 1998 by researchers Fire and Mello, this topic has been the focus of much research in areas such as biomedical research, health care, and even agriculture. Double stranded RNA has been found to play a crucial role in things such as pest control, vector borne disease prevention, crop improvement, and in the development of therapeutics for different diseases through gene silencing. Although much research has been focused on the effects of gene silencing, there is still much more needing to be done.

When scientists know what gene they want to manipulate they 'introduce double stranded DNA, or dsRNA to the cell.' The dsRNA produce small double-stranded interfering RNAs, or siRNA into the cytoplasm of the cell.5 from this the expression of the gene decreases drastically but does not get entirely eliminated, therefore showing the role of the targeted gene.

The Cas9 System and a small guide RNA molecule. The Cas9 is an enzyme that “snips through DNA like a pair of molecule scissors”(Zagorulya). The second component is a RNA molecule that acts as a guide to direct the Cas9 to the targeted sequence of DNA in the genome. DNA repair mechanisms in the cell can silence the gene if cut. When adding a new DNA fragment to the Cas9-gRNA complex, repair machinery repair DNA by adding the new DNA where the enzyme cut, through the DNA. With this, a mutation can be introduced into the gene or a new gene can be introduced into the cell DNA. Scientists are able to manipulate DNA in numerous ways using the system and study the function of health and mutated forms of specific genes. The flexibility of this system allows scientists to study any gene despite its location or composition of DNA. Flexibility is due to the RNAs design as it is created to target any site of the

There are varies of gene silencing methods and most of these methods involve disabling the function of mRNA by preventing it from being translated into a protein. However different methods use different design of molecules to disrupt mRNA. The most leading method of gene silencing is RNA interference

Next, crRNA biogenesis takes place in two stages. First, the CRISPR array and the Cas gene are transcribed respectively into a single pre-crRNA and Cas proteins. In this process, different types of CRISPR systems are unique in their Cas proteins they encode. Specifically, type II

1998). Interestingly, the phenomenon that the introduction of double-stranded RNAs “quelled” the expression of endogenous genes contain homologous sequences had been observed and even utilized as a tool to knock down gene expression by researchers long before the underlying RNAi mechanism was identified and separated from the antisense silencing by Fire and Mello in 1998 (Fire et al. 1998). They consistently found that the introduction of double-stranded RNAs (dsRNAs) was 100 or more folds effective than the single-stranded “sense” or “antisense” RNAs (ssRNA) to silence the complement gene expression (Fire et al. 1998; Kennerdell and Carthew 1998). Moreover, the dsRNA mediated gene silencing can be transferred between adjacent cells, and even inherited through multiple rounds of cell division (Fire et al. 1998). Therefore, it is proposed that this so called “RNA interference” is an inheritable and transferrable process that involves a catalytic process, which differs from the previously known antisense silencing that ssRNAs directly bind to the complement mRNA targets to repress translation by blocking ribosome access. Subsequently, Tuschl and co-workers demonstrated that RNAi mechanism exists in mammalian cell lines for the introduction of synthetic

In Elbashir, Lendeckel, and Tuschl’s article, they explore how 21- and 22-nucleotides RNAs mediate RNA interference by using an in vitro Drosophila system; this article is still referenced today as an authoritative source for RNAi research They show that the

Briefly, cells will be treated with cyclohexamide to arrest translating ribosomes. Extracts from these cells will be treated with RNase I to degrade regions of mRNAs not protected by ribosomes. The resulting 80S monosomes, which contain a ∼30-nucleotide RPF, will be purified on sucrose gradients and then treated to release the RPFs, which are then processed for Illumina high-throughput sequencing. In parallel, poly (A)-selected mRNA from each sample was randomly fragmented, and the resulting mRNA fragments will be processed for sequencing (mRNA-Seq) using the same protocol as that used for the RPFs. In summary, these experiments will pinpoint the targets, which are translated in UPF1 mutant cells.

Each repeat comprised of 33 to 34 amino acids. Different repeats vary mostly at position 12 and 13. Amino acids present at these two positions are called repeat variable di-residues (RVDs). Each RVD has preference for a nucleotide. Repeats are modular and can be assembled in required way to bind with any target DNA sequence (Gaber et al, 2014; Meckler et al, 2013). TALEs are thus a powerful and modular tool, which can be engineered to target any DNA sequence. Riboswitches are non-protein coding regulatory RNA, present in 5´ untranslated (UTR) region of mRNA, that upon binding with small molecules or peptides undergo conformational changes to control gene expression at translational level. Riboswitches are conceptually comprised of two parts (i) ligand specific aptamer domain, and (ii) expression platform, which undergoes structural changes in response to the changes in the aptamer (Winkler & Breaker, 2005). Ligand driven conformational change regulates translation either by sequestering ribosome binding site or by releasing it (Caron et al, 2012). Engineered riboswitches have been reported to be modular and work in dose dependent manner (Ceres et al, 2013; Dixon et al, 2010).

RISC in association with miR binds to 3’ untranslated region (UTR) of target gene mRNA to inhibit protein translation or promote RNA degradation.

Discovered in 1998 by Fire, Mello and colleagues, short interfering RNA (siRNA) is a small strand of

H-NS is known as a global regulator which influence the expression of large number of genes in response to various environmental factors like pH, osmolarity or temperature (Atlung and Ingmer, 1997; Dorman, 2007).Numerous studies can have shown the gene regulatory functions of H-NS in E.coli and Salmonella, acting mainly as a ‘gene silencer’(Dorman, 2004; Lucchini et al., 2006).Transcriptomic studies have revealed that H-NS regulates around 1439 genes in S.typhimurium. In uropathogenic E.coli strain, 536, H-NS regulates around 500 genes, including many virulence factors like cytotoxins, fimbriae and siderophores (Muller et al., 2006).H-NS is also responsible for the silencing of horizontally acquired DNA

About 1 % of the genome encodes for miRNA, and it is estimated that about one third of all human genes might be targeted by miRNAs. MiRNAs are transcribed from either intronic (coding and non-coding), or exonic regions of the genome. The primary transcription is typically several kilobases long with (a) hairpin structure(s) that contain(s) either one miRNA or a cluster of miRNA hairpin structures, as shown in Figure 1[31, 36, 37].

The miRNAs control gene expression post-transcription through different mechanisms such as repression at the initiation step by repression by preventing 60S subunit joining, deadenylating, proteolysis, and slow elongation or ribosome drop-off. This mechanism regulates mRNA translation or stability in the cytoplasm via non-perfect pairing with target mRNAs. Usually involving a seed pairing of just six to eight nucleotides in length cause degradation of target RNAs by the RISC complex in the case of perfect complementarity with the target site—the phenomenon known as RNAi. It is estimated that approximately one-third of human protein-coding genes are controlled by miRNA.