Group 2 Project - RNA Interference

From CellBiology
Student-drawn diagram 1. Summary of RNAi Process Diagrammatical representation of the cellular mechanisms of the RNA interference process

What is RNA Interference

RNA interference (RNAi) - also known as post-transcriptional gene silencing, RNA silencing or quelling - is an organisms response to being infected with exogenous genetic material whether it be DNA or RNA. This mechanism allows the organism to interfere with the foreign genetic sequence before it is able to integrate itself into the host genome and cause damage. Recently, this mechanism has been extensively used in several fields of research as it is effectively able to silence or "turn off" specific genes.

The marked difference between this technique, and that of "gene knockout", is that the gene remains present and completely unmodified, the only true effect is in the prevention of translation of messenger RNA (mRNA). During knockout, it is the gene itself that is acted upon.

RNAi is a particularly effective biotechnological research technique for identifying the functions of genes. [1] RNAi causes the silencing of gene expression within the cell by introducing double stranded RNA which is homologous to the target site of the gene. The gene silencing generally results not only from the inhibition of transcription through cleavage and degradation of target mRNA, but it also results from inhibition of protein synthesis by blocking translation of the intact mRNA. [2] RNAi is a powerful tool because of its specificity, efficiency and potency to knock down any specific gene of interest. [3] Two types of RNA molecules are utilised in RNAi - exogenously induced small interfering RNAs (siRNAs)and micro RNAs (miRNAs), which help regulate gene expression. [4]

History of RNAi

The timeline of RNA interference is widely accepted to be as follows - [5][6]

Petunia phenotype (wildtype vs RNAi).
The effects of RNAi on the phenotype of a petunia.

1990 Napoli C, Lemieux C, Jorgensen R. observed the phenomenon of mutual inhibition between the endogenous and transgenic mRNA from there experiment creating petunias with a deeper purple hue. This phenomenon of co-suppression was termed post-transcriptional gene silencing (PTGS). [7]

1995 Guo S, and Kemphues K J noticed that anti-sense RNA was effective in suppressing par-1 gene expression on C. elegans. They injected anti-sense RNA into the organism to cause gene silencing. Although their negative control also showed mediated gene suppression, it was still a remarkable discovery. [8]

1998 Fire et al. described the RNAi process in C. elegans. They used not only sense or anti-sense RNA, but also with double-stranded RNA (dsRNA)to inhibit gene expression in C. elegans. This experiment showed tenfold more effective geen silencing result than the result of with only sense or anti-sense RNA. This process was given the term RNA Interference. [9]

2000 Zamore et al. discovered the process of Dicer enzyme in cutting long dsRNA into shorter fragments in Drosophila. [10]

2001 Bernstein et al. was able to successfully clone Dicer enzyme. [11]

2001 Elbashir S M et al. were first to describe the presence of RNAi in mammalian cells. It was hard to achieve RNAi in mammalian cells because of the powerful interferon response induced by dsRNA which leads to the inhibition of all gene expression or cell death. Finally they achieved specific sequence of mRNA degradation without inducing the strong interderon response by introducing double stranded form of 21 to 22 nucleotide short interfering RNA (siRNA) duplexes. These studies now provides as a powerful tool in mammalian systems to selectively and rapidly suppress genes of interest. [12]

Fire and Mellow.
2006 Nobel Prize winners Andrew Fire and Craig Mello for their discovery of the RNAi mechanism.

2002 Jiang M, Milner J. demonstrated silensing of specific gene expression, viral E6 and E7 genes, by using siRNA in human papillomavirus (HPV) infected human cervical carcinoma cells. They showed the therapeutic potential for those diseases which caused by viral infection. [13]

2002 McCaffrey et al. showed the inhibition of transgene expression in adult mice by using siRNAs and small hairpin RNAs transcribed from DNA templates (miRNA). The possibility of therapeutic use of this technique was also mentioned by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo. [14]

2003 Paddison, Sui, Paul et al. noticed that Short hairpin RNA's act as dsRNA and can induce sequence specific silencing in mammals.

2003 Song et al. reported that small interfering RNA can be used therapeutically in mammals. [15]

2004 Morris et al. Observed that siRNA silences genes at the transcriptional level. [16]

2004 Acuity Pharmceuticals Clinical trial of siRNA drug for age related macular degeneration (AMD). [17]

2006 Andrew Fire and Craig Mello are awarded a Noble prize for discovering the mechanism of RNAi. [18]

2006 Li et al. Reported that dsRNA can induce gene expression and activation in human cells. [19]

Mechanisms of RNAi

Student-drawn diagram 2. Introduction and Cleavage of dsRNA

Introduction of Double-stranded RNA

The RNA interference (RNAi) process begins with the presence of a Double-Stranded RNA (dsRNA) molecule in a cell. This dsRNA can either be exogenous (introduced to the organism) or endogenous (produced by the cell itself).[20] Exogenous dsRNA can be introduced into an organism through:

  • Transfection - involves opening the pores of a cell membrane through electroporation or calcium phosphate and allowing the uptake of dsRNA.
  • Transduction - utilises viral vectors to administer the dsRNA into the target organism.

Endogenous dsRNA can appear in a cell as pre-microRNA which has a stem-loop structure.[21]

Cleavage of dsRNA

The presence of dsRNA is unusual within a normal cell. Whilst it is in the cell, it activates a protein endoribonuclease called Dicer. [22]Using ATP, the dicer protein is able to recognise the dsRNA and bind tightly to it. It then actively cleaves the long strand of dsRNA into smaller segments made up of 20-25 base pairs with overhanging ends.[23] Research suggests that this length maximises gene specificity by binding only to target sequences of that correspond to the RNA whilst minimising off-target effects which are corresponding RNA in an off target gene. These smaller segments are referred to as small interfering RNAs (siRNA).

Formation and activation of RISC

Student-drawn diagram 3. Formation and Activation of RISC, Gene silencing

One strand of the siRNA then binds to an argonaute protein in the cytoplasm, which is part of a multiprotein complex called RNA induced silencing complex (RISC).[24] The strand chosen for binding to RISC is the strand with the least thermodynamic stability on the 5’ end.[25] RISC is then activated when double-stranded siRNA is unzipped with the aid of ATP by the helicase protein of RISC. The guide strand is the RNA that is part of RISC complex and is involved in gene silencing, whilst the anti-guide strand (or passenger) is degraded during RISC activation.

Gene Silencing

Once activated, RISC recognises and binds to its complementary mRNA.If there is extensive binding to the mRNA, the argonaute component of RISC acts as an endonuclease and cleaves the target mRNA into two. The RISC complex is then released from the strand with the help of ATP, and other cytoplasmic proteins rapidly degrade the cleaved mRNA preventing its translation into a protein effectively silencing the gene, as well as inhibiting ribosomal attachment so as to block the translation process. [26]If there is less extensive binding, then the mRNA is transferred into cytoplasmic regions called P-bodies where it is eventually degraded resulting in reduced expression of the gene.

siRNA Design

In the laboratory RNAi usually starts with the introduction of exogenous Hairpin RNA encoded with the target siRNA rather than dsRNA.[27] The siRNA sequence used in the lab must be designed in a way that will target the site of interest and reducing target mRNA levels by at least 50%. Whilst there is no clear-cut procedure in how siRNA sequences should be manufactured, there are a few parameters which should be adhered to for efficient results. Guidelines for siRNA design include:[28]

  • Sequences should be 50-100bp downstream from the start codon of the target gene
  • GC content of target sequence should be between 30%-50% as these are generally more active than high GC content genes.
  • Several siRNA sequences should also be taken at different positions that adhere to the aforementioned parameters, this is to increase the likelihood of gene knockout.

At present, siRNA is produced by several companies available to be purchased by laboratories.

Applications

RNAi is a powerful research tool as it can conclude the purpose of a gene by disrupting its transcription. Normally, gene silencing by RNAi is incomplete and significantly lowers mRNA transcription levels rather than having complete gene knockout. However, this outcome is more than satisfactory for determining gene function in fields such as medicine and biotechnology.

Medicine

RNAi technique has the potential that could be used as a clinical method to inhibit gene expression in many diseases. This technique has the great possibilities that can provide the accuracy and selectivity to reduce side effects associated with the drugs used while treating various diseases particularly for treating cancer, HIV, vascular diseases, HBV, neuro-degenerative disorders, malaria, metabolic diseases, and so on.[29]


RNAi in Cancer treatment

Effects of siRNA transfection on HeLa cells. Anti-lamin RNAi transfected cells show very low lamin A/C staining levels in their nucleus.

RNAi is used for cancer therapy to inhibit the tumor growth and therefore killing cancer cells by knocking out the specific gene expression, cell cycle gene or anti-apoptopic gene, within cancer cells. RNAi is selectively used to target a gene specifically involved in the growth or survival of the cancer cell to kill cancer cells without damaging other normal resident cells. Small interfering RNAs (siRNAs) with the specific gene can also be used to inhibit the tumor growth by transfection. [30]

Current researches at Abbott Laboratories in America showed that they estimated genes Ran and TPX2 that play a role in maintaining human tumor cell survival by using RNAi technique. These genes are highly associated with the cells that had been affected by oncogene, also known as K-Ras. Therefore, researches are performed to target these two genes for the cancer therapy.[31]


HIV

Although the gene expression of HIV is understood, no cure has been made for it. Thus researches for RNAi against HIV have been a priority in medical research.[32] RNAi mediated inhibition to HIV infected cells was demonstrated. This research demonstrated that there is a possibility of blocking entry and replication of HIV by suppressing the chemokine receptor gene of HIV-1 with RNAi. However, despite the possibility of RNAi mediated inhibition to HIV, the virus itself is a difficult target to inhibit due to the high viral mutation rate.[33] This leads to escaping from being targeted. Therefore, further research is required to prevent escaping or avoiding the viral mutation.


Vascular Diseases

RNAi can also give the possibility of reducing the damage of heart and brain tissues by inhibiting the process of atherosclerosis. By selectively suppressing gene of cell adhesion molecule production within the vascular endothelial cells, the recruitment of neutrophils and macrophages to the damaged endothelial site can be reduced. [34]


Neuro-degenerative disorders

The use for RNAi therapy in those prone to particular neuro-degenerative disorders can quite cleverly be developed, through the gene silencing effect. Current studiest are focusing on Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and spinocerebellar ataxia - due to the developmental nature of the diseases.

Patients of a known genetic background, with a predisposition for various diseases, can utilise this technique in order to silence the genes involved in the pathogenesis stages.

As currently the causes for many neuro-degenerative disorders remain unknown, the process of RNAi is a breakthrough in the development of treating a patient, before the incident of genetic change has occured.[35]


Hepatitis B virus

Several studies on mice have been undertaken, successfully showing the effect of RNAi on hepatitis B virus replication. By inserting HBV specific siRNA into HepG2.215 cells it reduces the levels of mRNA and HBV proteins produced by approximately 88%. [36]

The results are promising so far, and many studies have concluded that RNAi is capable of inhibiting hepatitis B virus replication and expression in vitro and in vivo - which will continue to drive the research direction for a new therapeutic strategy for hepatitis B virus infection. [37]


Biotechnology

Picture from Nature Journal.
Differences between Wild type and RNAi affected coffee leaves].

RNA Interference has also recently been used extensively in fields such as functional genomics, design of transgenic plants as well as in medicine.


Functional Genomics

The ability of RNAi to selectively silence genes of interest allows it to determine the function and regulation of the selected gene on a cellular and a whole-organ level. The RNAi libraries that could be constructed through RNAi allows the determination of interrelated pathways and signalling networks that occur in an organism.


Design of Transgenic plants

RNAi is an important technique in the development of Genetically Modified Food. Recent studies show that by knocking out the expression of enzymes involved in caffeine production in coffee leaves, it is able to produce transgenic decaffeinated coffee leaves.[38] Another study shows that RNAi has shown potential in being used to repress the expression of certain subunits of the Gliadin protein in wheat. This protein is responsible for Coeliac Disease (Gluten allergy) which is rapidly becoming a problem in todays community. These gene knockout studies on Gliadin can further enhance our knowledge on Coeliac disease as well as provide an alternative source of wheat.

Advantages & Disadvantages

RNA Interference use in Gene silencing
Advantages of RNAi Disadvantages of RNAi
This process is able to affect only selected genes which the RNAi is complementary to. There are off-target effects when RNAi is used. This is when siRNA can affect unintended genes in the organism which may be vital
RNAi will bind to most of the complementary genes it encounters, making it highly efficient as well as robust RNAi is still a relatively poorly understood process.
Shown to work in the laboratory and shows potential in mammalian cell The introduction of external RNA into a mammalian cells whether it be from a viral vector or through the chemical synthesis of a reagent can potentially induce an immune response such as interferon release which may be toxic to the host. This makes delivery to mammals a very difficult process
Able to be used on a large scale

Future perspectives

Since RNAi discovery in 1998, it has been touted as a technical breakthrough in biological research. It's ability to accurately silence selected genes in vivo and in vitro has had some scientists predicting that RNAi may even surpass PCR as the most important technological advancement. [39]

Even with RNAi's rapid development over the years, it is still in its infancy stage. A better understanding of the mechanisms that take place will help reduce problems such as off-target effects. The development of a more efficient delivery method, whether it be better quality invasive transfection reagents or in cell lines and the conditions of the culture, will also stop or limit the immune system from reacting to RNAi.

The future of RNAi is only beginning to be realised. In 2001 RNAi was used to treat hepatitis in mice, with further knowledge about the mechanisms of RNAi it may be the gateway for other emerging technologies such as transgenic studies, gene therapy and gene-wide screening.[40]

A current study (April 2010) from the University of Amsterdam is investigating a technique using RNAi in the therapy of patients with HIV, that continues to provide long-lasting effects even after a single treatment. [41]

In order to achieve this, antiviral DNA is delivered to extracted stem cells from the patient, before being re-injected within the body.

The DNA encodes RNA found within the immune cells, and allows them to pair up against viral genes when encountered - through the process of RNAi - blocking the production of viral components from the genes.

Whilst still in process, it opens the doors of what can be achieved, and infact realises a small part of the hope - that nothing is untreatable.

Glossary

Aforementioned parameters:

Argonaute protein: A protein present within the cell which is the catalytic components of the RNA induced silencing complex (RISC)

Atherosclerosis: Vascular disease characterized by irregularly distributed lipid deposits in the intima of large and medium sized arteries, causing narrowing of arterial lumens and proceeding eventually to fibrosis and calcification.

Dicer: A specific RNase III normally present within the cell

Downstream of RNA (or DNA) : The position on the RNA (or DNA) strand. Downstream is the region towards the 3' end of the strand and upstream is the region towards the 5’end.

dsRNA: Double-stranded RNA

Endogenous: Originating or produced within the organism or one of its parts.

Exogenous: Originating or produced outside of the organism.

Gene silencing: A specific gene is turned off by a mechanism in the cell other than genetic modification so that the gene expression does not occur for the specific site.

Helicase: An enzyme capable of unwinding and separate the DNA or double-stranded RNA strands by breaking hydrogen bonds in its path.

miRNA: Micro RNA

Overhanging end: A stretch of unpaired nucleotides in the end of a DNA/RNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs.

RISC: RNA induced silencing complex

RNAi: RNA(Ribonucleic acid) interference

siRNA: Small interfering RNA

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2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer