3. History of RNA Interference

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Paper No. : 15 Molecular Cell Biology

Module : 29 RNA mediated Mechanisms: RNA Interference

Development Team

Paper Coordinator: Prof. Kuldeep K. Sharma

Department of Zoology, University of Jammu i

Principal Investigator: Prof. Neeta Sehgal

Department of Zoology, University of Delhi

Content Writer: Dr. Nidhi Garg

Deshbandhu College, University of Delhi

Content Reviewer: Prof. Rup Lal

Department of Zoology, University of Delhi Co-Principal Investigator: Prof. D.K. Singh

Department of Zoology, University of Delhi

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Description of Module

Subject Name ZOOLOGY

Paper Name Molecular Cell Biology; Zool 015

Module Name/Title RNA mediated Mechanisms: RNA Interference Module Id 29; RNA Interference

Keywords RNAi, siRNA, miRNA, PTGS

1. Learning Outcomes

After studying this module, you shall be able to

• Know how RNA interference works.

• Learn about various mechanisms of RNA interference.

• Evaluate the importance of RNA interference in regulation of gene expression.

• Know the current and the potential applications of RNA interference.

2. Introduction

RNA interference (RNAi) is defined as a mechanism or a biological process where the RNA molecules interfere with the gene expression thereby inhibiting it, by destroying specific mRNA molecules. These RNA molecules for the purpose of inhibiting gene expression, share sequence homology with the gene in question. The RNA interference is mediated by a double-stranded RNA (dsRNA) which is clipped into small RNAs by the enzyme Dicer. These small RNAs associate with several silencing effector complexes which then attach to homologous target sequences which may be RNA or DNA by base pairing. Then on the basis of protein composition of the effector complex as well as the nature of the target sequence, these RNA molecules can lead to mRNA degradation, translational repression, or genome modification. All these three processes lead to silencing of the gene expression (Figure 1). The act of repressing both transcription and translation is known as RNA- mediated gene silencing.The RNA-mediated gene silencing pathways are known to exist in plants, animals, and several fungi, where they play important roles in development, chromosome structure, and virus resistance.

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Figure 1: Mechanisms of gene silencing in eukaryotes

Source: Matzke, M. A. and Matzke, A. J. M. 2004. Planting the Seeds of a New Paradigm. PLoS Biology. Vol. 2: 0582-0586.

The phenomenon of RNA interference was first discovered in plants in petunia. Initially, the mechanism of RNA interference was not well characterized and it was known as post transcriptional gene silencing (PTGS) and transgene silencing. Short RNA molecules, approximately 21 nucleotides long are known to regulate gene expression within in the cytoplasm of plants, animals and fungi. These RNA molecules work by repressing translation and eliciting the degradation of mRNAs.

Thus, RNA interference is a type of sequence-specific post-transcriptional regulation mechanism.

Certain short RNAs are capable of altering the chromatin structure thereby causing the repression of transcription.

3. History of RNA Interference

Several biologists came to know of RNA-mediated gene silencing in 1998 only after the discovery of RNA interference in Caenorhabditis elegans, where a dsRNA initiates the sequence-specific mRNA degradation. Whereas, the roots of RNA-mediated silencing, or RNA interference dates back to late 1980s, when botanists across the globe were struggling with strange cases of gene silencing in transgenic plants. The RNA molecules were commonly used for bringing about a reduction in the gene expression in plants for many years. The single-stranded antisense RNA molecules were

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introduced into plant cells which subsequently hybridized to the corresponding single-stranded, sense mRNA. In early 1990’s, while working with petunias, the researchers tried changing the flower colors. To achieve this, they injected multiple copies of chalcone synthase gene. The enzyme chalcone synthase is responsible for imparting pigmentation to the flowers of petunias, which are usually pink or violet colored. Contrary to the expectation of obtaining darker colored flowers, the overexpression of chalcone synthase gene instead resulted in less pigmented, fully or partially white flowers (Figure 2). This simply meant a substantial reduction in the activity of chalcone synthase. In fact, these white flowers were a result of down regulation of both the endogenous genes and the transgenes.

Figure 2: Examples of petunia plants where genes responsible for pigmentation were silenced by RNA interference. The left petunia plant is wild-type. The petunia plants to the right contain transgenes which have resulted in unpigmented white areas of the flower

Source: https://en.wikipedia.org/wiki/File:Rnai_phenotype_petunia_crop.png

Researchers upon further investigation found that the down regulation of chalcone synthase gene was caused by post-transcriptional inhibition of gene expression or PTGS due to high level degradation of mRNA. This was known as co-suppression of gene expression, but the underlying mechanism responsible for it was not known.A related phenomenon known as “quelling”, was observed in the filamentous fungus Neurospora crassa but the details were not worked out. In 1994, the phenomenon of RNA-directed DNA methylation was discovered in transgenic tobacco plants which happened to be the first reported case of RNA-induced modification of DNA. The next development was that plant RNA viruses could be both initiators and targets of post transcriptional gene silencing (PTGS).

Researchers were working to increase plant resistance towards viral diseases. Plants which expressed virus-specific proteins exhibited greater resistance to viral infection was known. But it was unknown, that this phenomenon was exhibited by plants having only short, non-coding regions of viral RNA sequences. Researchers believed that the viral replication was probably inhibited by the viral RNA produced by the transgenes. In an experiment, when short sequences of plant genes were introduced in viruses, it led to suppression of the target gene expression in an infected plant. This phenomenon was called as "virus-induced gene silencing" (VIGS). This occurred due to the mutual degradation of

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viral RNA and transgene mRNA. This study not only established a link between RNA virus resistance and PTGS, but also discovered the role of RNA dependent RNA polymerase (RDR) which can synthesize dsRNA from ssRNA templates, small RNAs, and dsRNA in the process. All these were later found to be mediators of RNAi. In 1997, PTGS was reported to protect plants naturally from virus infection.

Thus, by the year 1998 when RNAi was reported the plant scientists had already documented the following:

1. Sequence-specific RNA degradation (PTGS)

2. Sequence specific DNA methylation that triggered TGS 3. RNA-directed DNA methylation

4. Proposed models for PTGS involving dsRNA

After working with plants, the researchers set out to look for similar phenomenon in other organisms as well. An outcome of this was the discovery of the phenomenon of RNA interference by Craig C.

Mello and Andrew Fire for which in 2006 they were awarded the Nobel Prize in Physiology or Medicine. They coined the term RNAi and their pioneering work was published in the journal Nature1998. The experiments involved comparing the silencing activity of antisense or sense single- stranded RNAs (ssRNAs) and the dsRNA hybrid. The nematode worm C. elegans was injected with both types of RNA molecules. But only low level of gene silencing was achieved with ssRNAs, while injection of a dsRNA hybrid resulted in potent and specific silencing. This led to establishment of dsRNA molecules as the trigger of silencing. Their research opened up a new branch of molecular biology, with would have far-reaching implications because of their practical applications.

Shortly after the discovery of RNAi, in 1998 itself dsRNA was shown to cause gene silencing in other animals and plants. In 1999, scientists studying PTGS in transgenic plants detected the small 25 nucleotide-long RNAs involved in mediating gene silencing. In 2000-2001, researchers working with Drosophila demonstrated short interfering RNAs known as siRNAs, that are 21–23 nucleotides long, are derived from cutting of longer dsRNA, were capable of guiding mRNA cleavage. In 2000 itself, the nuclease known as RNA induced silencing complex (RISC), was discovered which associates itself with small RNAs and performs cleavage of target mRNA. In the year 2001, the enzyme known as Dicer was identified which cuts dsRNA into short RNAs. In 2001, through cloning several natural small RNAs 21–25 nucleotides long, were isolated first from worms and flies and then from plants and mammals. Small sized RNAs known as microRNAs (miRNAs), were identified in 2003, which

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arise from Dicer processing of dsRNA which are then incorporated into RISC. miRNAs are the key regulators of plant and animal development.

In 2003, the relatedness of RNAi, PTGS, and quelling was established through genetic analyses in worms, plants, and Neurospora which led to the identification of common components involved in the respective silencing pathways. PTGS is now believed as equivalent of RNAi in the plant system. The next major advancement came in 2002, with the discovery of RNAi mediated heterchromatin assembly in fission yeast, which established the principle of RNA-guided genome modifications. This pathway involves the use of short RNAs produced by Dicer and other RNAi components for methylating histones. This generates condensed, transcriptionally inactive regions called as heterochromatin such as centromeres. In 2003, RNAi-dependent heterochromatin pathway was discovered in plants and in 2004 in Drosophila. Thus, RNAi plays an important role in creating condensed, silent chromosome domains.

4. Craig Mello and Andrew Fire’s Classic Experiment

They chose the model organism roundworm Caenorhabditis elegans for their work. In order to test the possibility of interference by double-stranded RNA molecules, they purified single-stranded RNA molecules. Next, they compared the interference level between the individual strands verses the double-stranded hybrids. The unc-22 gene was the gene of choice for the experiment which encodes an abundant but nonessential myofilament protein. Within each striated muscle cell thousands of copies of unc-22mRNA were present. They injected C. elegans cells with either single-stranded or double-stranded RNA molecules that shared sequences homology with the unc-22 gene’s mRNA. The expected outcome was the down regulation of unc-22 gene expression by the single-stranded antisense RNA molecules which would simply bind to the endogenous sense mRNA. But to their surprise the double-stranded unc-22 RNA was about 10- to 100-fold efficient in repressing theunc-22 mRNA expression. A decreases in the expression of unc-22 leads to an “increasingly severe twitching phenotype”, while a complete loss of function leads to “muscle structural defects and impaired motility”.

Injection of unc-22 ssRNA into adult animals produced a modest effect, while unc-22 dsRNA produced potent and specific interference and phenotype. Both the injected animals and their progeny showed behaviour similar to loss-of-function mutations in unc-22. The presence of dsRNA initiates the degradation of the mRNA provided the mRNA has a sequence complementary to one of the

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strands of the dsRNA. Few molecules of dsRNA are required to initiate degradation of large amounts of mRNA.

Thus, with this experiment the dsRNA became a new tool for studying the gene function in not only C. elegans but also in a number of other organisms. Even though the introduction of dsRNA produced interference that was potent and specific there are several limitations in designing RNA-interference- based experiments.

1. When sequence homology exists between several closely related genes introduction of dsRNA molecules may interfere with the functioning of several members of the gene family.

2. There exists a possibility of low level of expression that will resist RNA-mediated interference for either some or all genes.

3. It is quite likely that small number of cells might escape the effects of RNA-mediated interference.

5. Molecular Mechanisms of RNA Induced Gene Silencing

The nature has evolved an efficient mechanism for gene silencing through the use of small 21-25 nucleotide long, non-coding RNAs that are double stranded and bind to the target RNAs in a sequence-specific manner. There are three main classes of small RNA namely

1. Short interfering RNAs (siRNAs) 2. MicroRNAs (miRNAs)

3. PIWI-interacting RNAs (piRNAs)

Although these three RNA’s arise from different sources, their mechanisms of action are similar.

5.1. Short interfering RNAs (siRNAs)

They are derived from longer RNA molecules which are linear, double stranded and located in the cell cytoplasm. siRNA precursors arise within cells from-

 RNA virus replication

 Transcription of cellular genes

 Self-annealing transcripts

 Expression of transposons

 Experimental transfection

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The siRNAs, mediate RNA interference by slicing or the endonucleolytic cleavage of their target RNAs. RNAi may be a mechanism through which cells recognize the viral RNAs and inactivate them, thereby protecting the organism from either external or internal assaults. Within the cells cytoplasm, the endonuclease Dicer recognizes these double stranded RNA molecules and then cleave them into siRNAs of length approximately 21–25-nucleotide,which at its 3` end contains a 2-nucleotide overhang. Comparative genomics suggests that, this length helps in maximizing the target-gene specificity while subsequently minimizing the non-specific effects.

Figure 3: The 3-dimentional structure of dicer protein isolated from Giardia intestinalis, that catalyzes the cleavage of dsRNA to siRNAs. The RNase domains are colored green, the PAZ domain yellow, the platform domain red, and the connector helix blue.

Source: https://en.wikipedia.org/wiki/File:2ffl-by-domain.png

Next one of the strand of the siRNA duplex known as the guide strand is loaded onto an Argonaute protein. The Argonaute protein is at the core of an RNA-induced silencing complex (RISC). RISC is a ternary complex which comprises of three main components namely

1. An Argonaute protein 2. Dicer

3. A dsRNA-binding protein known as TRBP in humans

The second strand of the RNA known as the non-guide or passenger strand during the process of loading is cleaved by an Argonaute protein. The guide siRNA is used by the Argonaute protein for associating with the target RNAs containing perfectly complementary sequence which are

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subsequently sliced. The cleaved target RNA after slicing is released, while the RISC is recycled for the next round of slicing (Figure 4).Both the enzymes Drosha and Dicer belong to the RNase III family which upon cleavage of dsRNA produce small RNA molecules with termini containing a monophosphate group at the 5ʹ end, and a two-nucleotide overhang at the 3ʹ end.

Figure 4: Mechanism of action of siRNA

Source: Jinek, M., and Doudna, J. A. 2009. A three-dimensional view of the molecular machinery of RNA interference. Nature. Vol 457: 405-412.

5.2. MicroRNAs (miRNAs)

MicroRNAs are short RNA molecules transcribed from the cells own DNA within the nucleus, but are never translated into proteins. The DNA sequence that codes for miRNA gene is longer than the miRNA itself as it includes miRNA sequence and its reverse complement. The miRNA sequence and its reverse-complement upon transcription base pair giving rise to double stranded stem–loop structures called primary miRNA structure (pri-miRNA) that are 65–70-nucleotide long (Figure 5).

These RNAs are transcribed from two sources-

 Sequences within the introns of other protein-coding genes

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 From their own promoters

The human genome has more than 700 noncoding RNA genes, Arabidopsis has more than 130 while C. elegans has more than 100 noncoding RNA genes. Their main function in the eukaryotic genome is to regulate the expression of majority of protein-coding genes. According to an estimate about 30% of human genes are regulated through miRNA-related mechanisms.

Figure 5: Pathway involved in the production of miRNA

Source: Corradini, R., Manicardi, A., Sforza, S., Tedeschi, T., Fabbri, E., Borgatti, M., Bianchi, N., and Gambari, R. 2011. Gene Modulation by Peptide Nucleic Acids (PNAs) Targeting microRNAs (miRs). Targets in Gene Therapy. ISBN 978-953-307-540-2.

The enzyme complex known as Drosha–DGCR8 complex is localized within the nucleus recognize these hairpin stem–loop structures to excise it yielding a pre-miRNA molecule. Drosha is an RNase III enzyme while DGCR8 is a dsRNA-binding protein. Next the pre-miRNA molecule is exported from nucleus into cytoplasm by Exportin 5, a carrier protein where the enzyme Dicer cleaves the pre- miRNA, 20-25 nucleotides from the base of the hairpin giving rise to a short, linear miRNA–miRNA*

duplex (where miRNA denotes the guide strand and miRNA* the passenger strand). In plants, that do not express Drosha, the processing pri- miRNA and pre-miRNA is done by Dicer probably within nucleus and the mature miRNA duplexes are then exported to the cytoplasm by the Exportin 5. The plant miRNAs mediate the slicing of target mRNAs just like siRNAs, whereas the animal miRNAs silence target mRNAs without slicing (Figure 6). The guide strand is loaded onto an Argonaute

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protein just like in siRNAs. In case of animal systems, the miRNAs have been reported to be only partly complementary to sequences of their target mRNAs 3' untranslated regions. Due to partial complementarity the Argonaute protein does not slice the target mRNA. Moreover, certain Argonaute proteins lack the catalytic residues required for slicing the mRNAs. It is perhaps the guide strand’s 5’

end that performs the dual function of matching and binding to the target mRNA, whereas the 3’ end helps to position the target mRNA into the RISC region responsible for cleavage. The complete mechanism of miRNA-mediated silencing is still to be worked out but have been proposed to take place by repressing the translation of target mRNA and removal of mRNA poly(A) tails which cause the degradation of mRNA. The Argonaute proteins are found in specific areas within the cytoplasm known as P-bodies also known as cytoplasmic bodies or GW bodies. The P-bodies are regions characterized by high rates of mRNA decay as well as miRNA activity. The disruption of P-bodies leads to a decrease in the efficiency of RNA interference, thereby signifying its critical role in the RNAi process.

The gene silencing machinery is used by the cell for the regulation of gene activity. Sometimes some portions of the genome are transcribed into microRNA, having a hairpin shape. The RNA interference machinery upon detection of these double strands will automatically initiate the destruction of all mRNAs similar to microRNA. This prevents their translation and lowers the activity of several other genes.

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Figure 6: Mechanism of action of miRNA

Source: Jinek, M., and Doudna, J. A. 2009. A three-dimensional view of the molecular machinery of RNA interference. Nature. Vol 457: 405-412.

5.3. PIWI - interacting RNAs (piRNAs)

The third type of molecules mediating RNA interference is the piRNAs that are approximately 24–31 nucleotides long. Their main function is to silence transposons in the germ cells of animals. Currently, both the biogenesis as well as the mechanism of action of piRNAs is not well understood. Some understanding of this model has been worked in Drosophila melanogaster (Figure 7). Researchers have discovered ssRNAs as the precursor molecules of piRNAs. The enzyme Dicer is not required during the biogenesis of piRNA . The piRNAs cause slicer-dependent cleavages of both sense and antisense transposon transcripts which is facilitated by the proteins of the Argonaute family, such as PIWI, Aubergine (AUB) and Argonaute 3 (AGO3) in D. melanogaster. The sense piRNAs are generated via slicing of sense transcripts by PIWI or AUB proteins. The piRNAs then associate with AGO3 to direct the slicing of antisense transposon transcripts. This generates antisense piRNAs,

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which in then bind to PIWI and AUB proteins in order to guide the slicing of sense transposon transcripts for generating the sense piRNAs.

Figure 7: Mechanism of action of piRNA

Source: Jinek, M., and Doudna, J. A. 2009. A three-dimensional view of the molecular machinery of RNA interference. Nature. Vol 457: 405-412.

6. Pathways involved in RNA Induced Gene Silencing

The RNA interference pathway takes several steps (Figure 8).

1. First of all, either the siRNA or the miRNA molecules associate with the RNA-induced silencing complex (RISC).

2. Secondly, within RISC, the short double-stranded RNA is denatured followed by the degradation of the sense strand.

3. Thirdly, the RNA/RISC complex becomes fully functional and highly specific by finding mRNA molecules that have a sequence complementary to the antisense RNA present in the RISC.

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4. At this juncture RNAi pathway proceeds in two different directions depending upon the complementarity of target mRNA to the antisense mRNA present in the RISC.

5. If the sequence of the target mRNA is perfectly complementary to antisense RNA in the RISC then the RISC will cleave the mRNA which is subsequently degraded by ribonucleases.

6. But if sequence of the target mRNA is not exactly complementary to that of the antisense RNA within the RISC then the RISC complex binds to the mRNA, which represses its translation.

7. RNAi is a powerful biological mechanism for silencing the gene expression by either affecting the stability or the translation of mRNA; heterochromatinization of the genome leading to repression of gene transcription. These functions are performed by associating with the RNA-induced initiation of transcription silencing complex (RITS).

8. The antisense RNA strand within the RITS directs the RITS complex to definite gene promoters or to the larger regions of chromatin. The function of RITS is torecruit the chromatin remodelling enzymes to these genomic regions which in turn methylate histones and DNA, causing heterochromatin formation and subsequent transcriptional silencing.

Figure 8: Gene regulatory mechanisms by RNA induced gene silencing

Source: Klug, W. S., and Cummings, M. R. 2010. Concepts of Genetics. 10^th Edition. Pearson Education Pte. Ltd.

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7. Biological Functions of RNA Interference

7.1. Immunity

RNAi is an important component of the immune response when either viruses or any foreign genetic material, enters the body of plants and animals. In plants RNAi prevents the self-propagation of transposons also. Arabidopsis thaliana has been reported to express several homologs of the enzyme dicer each of which is specialized to react differently against different viruses. Certain plants upon infection by particular bacteria express endogenous siRNAs. In Drosophila, RNAi provides antiviral innate immunity and also protects actively against pathogens like Drosophila X virus. In C. elegans, the argonaute proteins and other components of the RNAi pathways are up regulated upon viral infection thereby conferring resistance to the worm. As of now the RNAi exact role in mammalian innate immunity is not well understood.

7.2. Down regulation of genes

It was in 1993, when the researchers first reported in C. elegans the function of miRNA in down regulating the expression of gene. Similar function is performed by the JAW microRNA in plant Arabidopsis were it regulates several genes that control plant shape. Both intronic and intergenic miRNAs, mediate translational repression, regulate development particularly the timing of morphogenesis and maintains undifferentiated cell types like stem cells. miRNAs are involved in epigenetic phenomena such as gene imprinting and X-chromosome inactivation. RNAi is responsible for the formation of centromeric structure in chromosomes. RNAi pathways repress transcription in two ways-

 By targeting transcription factor mRNAs

 Reduction in the levels of specific transcription factors automatically reduces transcription of genes.

In plants, the miRNAs mainly regulate the genes encoding the transcription factors and the F-box proteins. In abnormal functioning of miRNAs are associated with the occurrence of diabetes, heart disease, tumor formation and imbalance of the cell cycle. Thus, miRNAs perform the dual role of oncogenes and tumor suppressors.

7.3. Up regulation of genes

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Both siRNA and miRNA play an important role in increasing gene expression. This is because they have f sequences that are complementary to the sequence of the target gene’s promoter which can escalate gene transcription. This is known as RNA activation. This occurs with the help of dicer and argonaute proteins which possibly cause histone demethylation.

8. Applications of RNA Interference

8.1. Biotechnology

RNAi has several applications in biotechnology as mentioned below.

8.1.1. Gene knockdown

RNAi-mediated gene silencing is a comparatively specific and inexpensive method that allows scientists to rapidly analyse the functions of a gene either in cell culture or in model organisms. Ds RNA molecules having sequence complementary to the target gene are synthesized and introduced into a cell or organism. Once inside the biological system the dsRNA molecules are considered to be foreign genetic material thereby activating the RNAi pathway which causes a severe reduction in the target gene’s expression. RNAi is also known as "gene knockdown", because it does not completely abolish gene expression unlike the "gene knockout" procedures where gene expression is totally eliminated.

The biggest advantage of using RNAi is that it helps in creating specific single-gene defects without the need to create a transgenic organism carrying the mutation in its genome. Synthetic siRNA molecules are commercially available to be used in research. Using computational biology researchers are trying to design ds RNA molecules which will maximize the process of gene knockdown while simultaneously minimizing the off-target effects”. Off-target effects occur due to pairing of the introduced RNA with multiple mRNAs thereby reducing the expression of multiple genes. This usually occurs due to the presence of repetitive sequences in the dsRNA. The exogenous RNA to be introduced can be either long so it is cleaved by dicer, or short so it serves as siRNA substrates based on the organism and experimental system. In mammalian cells, siRNAs are used because long dsRNA molecules activate interferon response, which causes non-specific reaction to exogenous genetic material. Either oocytes or early embryo cells of mouse are used as mammalian model system for studying gene-knockdown as they do not show interferon response to exogenous dsRNA.

The applications of RNAi in crop science have far reaching consequences. Using RNAi, the researchers have developed nicotine free tobacco, decaffeinated coffee, nutrient fortified and

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hypoallergenic crops. In the Arctic apples polyphenol oxidase gene (PPO) has been suppressed through RNAi. This will prevent the apple from undergoing browning once it is sliced as there will be no conversion of chlorogenic acid into quinone. These apples will soon be receiving US approval.

RNAi has been used in plants to lower the levels of natural plant toxins. An example of which are the cotton seeds which contain the toxic compound gossypol that makes them unsuitable for human consumption. Through the use of RNAi, the levels of enzyme delta-cadinene synthase which is involved in gossypol production has been reduced in the cotton seeds. But the production of this enzyme is unaffected in other parts of the plant, as gossypol protects the plant from damage by pests.

The technique of RNAi has been used for reduction of the linamarin in cassava plants. Through the use of RNAi researchers there has been significant reduction in allergen levels in tomato plants. RNAi has been successfully used in the laboratory to develop resistance to common virus affecting plants.

8.1.2. Transgenic plants

Transgenic crops expressing RNA, to silence important genes in target pests have been developed.

Monsanto will be marketing the transgenic corn seed expressing dsRNA.The dsRNA has been derived from the gene Snf7and this gene was discovered in a beetle called the western corn rootworm. Every year this beetal’s larvae cause damage worth one billion US dollars in the USA. A study conducted in 2012 showed that silencing of Snf7 gene results in stunted larval growth, which kills them within days. Later in 2013 the same team reported negligible effects of RNA effects on other species. Effort are being made by researchers to introduce gene silencing in other organisms like catterpillars, ants, and in pollen beetles.

8.1.3. Insecticide

RNAi is being developed as an insecticide. In order to delay the development of resistance towards insects the researchers are trying to incorporate the protein Cry, and RNAi in one plant. As of now not much success has been achieved as there is no negative effect of RNAi on insects such as the cotton bollworm, the beet armyworm and the Asiatic rice borer. Use of RNAi in different species of butterflies and moths gave varying results as the RNA is effectively broken down by their saliva.

8.2. Medicine

Research is on to develop RNAi as a potential pharmaceutical agent against a disease caused either by overexpression of a gene or normal expression of mutant gene. The in vivo delivery of RNAi is without difficulty reachable to surface tissues like the eye and respiratory tract. For example, when the siRNA was kept in direct contact with the tissue it effectively focused on the targeted genes.

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Protection from nucleases is necessary to deliver siRNA to deep tissues, otherwise targeting specific areas is problematic. To overcome this high levels of siRNA are injected to ensure their delivery to tissues which in some cases led to hepatotoxicity. Surface application of siRNA molecules in animal models; have proved successful in treating virus infections, eye diseases, cancers and inflammatory bowel disease. Application of RNAi for the potential treatments of neurodegenerative diseases like the Huntington's disease has been proposed.

8.2.1. Antiviral RNAi therapy

Using RNAi in tissue cultures there was a reduction in the severity of infection by HIV, influenza and polio virus. Potential antiviral therapies include

1. Treating infection by herpes simplex virus type 2

2. In cancerous cells, there is inhibition of viral gene expression 3. Knockdown of both the host receptors and the co-receptors for HIV 4. Silencing of influenza genes, as well as hepatitis A and B genes 5. Inhibition of replication of measles viral

8.2.2. Cancer

RNAi technology can be applied for treating cancers by silencing overexpression or abnormal expression of key proto-oncogenes in tumor cells. Different types of cancers have differential expression profiles of miRNA which can be used for diagnosing tumors, predicting their course and planning of treatments. In some cancers there is defect in miRNA gene expression, which can be treated by injecting synthetic siRNAs. There is a need for the development of a safe delivery method for transporting RNA molecules. Presently viral vector systems are used but due to safety concerns non-viral delivery methods like liposomes are being considered.

A lot of progress has been made in developing RNAi pharmaceuticals-

 Completion of phase I (toxicity) of the first human clinical trials.

 Completion of phase II and III (efficacy) clinical trials for using siRNAs to treat age-related macular degeneration caused by expression of the vascular endothelial growth factor gene.

 Clinical trials to test the use of RNAi for treating Hepatitis B and respiratory syncytial virus infections have begun.

 Clinical trials are planned for using siRNA to treat influenza and hepatitis C virus infections and some solid tumors.

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 In mouse models RNAi has proved to so be effective that it has led toreversal of induced liver failure.

9. Summary

 RNAi is a phenomenon where the RNA molecules lead to gene silencing by initiating mRNA degradation, translational repression, or genome modification.

 RNA-mediated gene silencing was first discovered in plant petunia but the underlying mechanism was not known.

 It was Fire and Mello’s work which led to the discovery of RNAi in C. elegans. The RNA interference is mediated by a dsRNA that share sequence homology with the mRNA or the gene they target.

 RNAi is majorly mediated by siRNA and the miRNA.

 siRNA is derived from longer, linear, dsRNA molecules located in the cell cytoplasm. The enzyme Dicer cleaves the dsRNA into ~21–25-nucleotide long siRNAs with an overhang at the 3' end.

 The guide strand of siRNA is loaded onto an Argonaute protein which is the component of RNA- induced silencing complex (RISC) while the non-guide strand is cleaved by an Argonaute protein.

The guide siRNA associates with the target RNAs containing perfectly complementary sequence which are subsequently sliced.

 miRNAs are short RNA molecules transcribed from the sequences within the introns of other protein-coding genes or from their own promoters.

 The miRNA upon transcription has a double stranded stem–loop structures called primary miRNA structure (pri-miRNA) that are 65–70-nucleotide long which is subsequently processed to pre-miRNA by the Drosha–DGCR8 complex.

 The pre-miRNA is exported to cytoplasm by Exportin 5, where the enzyme Dicer cleaves it to 20- 25 nucleotides long linear ds-miRNA. The guide strand of miRNA is loaded onto an Argonaute protein of RISC. The miRNAs are partly complementary to sequences at the 3' untranslated regions of their target mRNAs hence the Argonaute protein does not slice the target mRNA.

 The piRNAs are the third class of RNA molecules mediating RNAi. They are ~24–31 nucleotides long and silence the transposons in animal germ cells.

 Biological functions of RNAi include protection against viral infections, upregulation and down regulation of genes.

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 Applications of RNAi are in the field of basic research, biotechnology, medicine, agriculture, functional genomics etc.

3. History of RNA Interference

Development Team

Contents

1. Learning Outcomes

2. Introduction

3. History of RNA Interference

4. Craig Mello and Andrew Fire’s Classic Experiment

5. Molecular Mechanisms of RNA Induced Gene Silencing

6. Pathways involved in RNA Induced Gene Silencing

7. Biological Functions of RNA Interference

8. Applications of RNA Interference

9. Summary