RNAi

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RNA interference

INTRODUCTION''

The understanding of how genes are turned on and off in a cell has been revolutionized by the discovery of RNA Interference. It has been heralded as a major scientific breakthrough that happens once every decade or so for which Fire and Mello , its discoverers were awarded the 2006 Nobel Prize for physiology. They received the prize for their discovery that dsRNA triggers suppression of gene activity in a homology dependent manner. Its discovery revealed a new mechanism for gene regulation that occurs in organisms ranging from plants to mammals. This system has proven to be important for both development of an organism and physiological function of the cells and tissues. Furthermore, RNAi protects against RNA virus infections especially in plants and invertebrates, and secures genome stability by keeping mobile elements silent. Researchers have quickly capitalized on this natural process and RNAi, now being considered as a powerful research tool, is used in thousands of labs worldwide.

Discovery of RNAi

Prior to the discovery of RNA interference, a phenomenon called gene silencing was described in plants. It was first noted in Petunia plants. In an attempt to alter flower colors in Petunias, researchers introduced additional copies of a gene encoding chalcone synthase, a key enzyme for flower pigmentation into Petunia plants of normally pink or violet flower color. The over expressed gene was expected to result in darker flowers, but instead produced less pigmented, fully or partially white flowers, indicating that the activity of chalcone synthase had been substantially decreased; in fact, both the endogenous genes and the transgenes were down regulated in the white flowers. Further investigation of the phenomenon in plants indicated that the down regulation was due to posttranscriptional inhibition of gene expression via an increased rate of mRNA degradation. This phenomenon was called cosuppression of gene expression, but the molecular mechanism remained unknown. Mello coined the term RNA interference for this unknown mechanism. Andrew Fire and Craig Mello published their breakthrough study on the mechanism of RNA interference in Nature in 1998.They tested the phenotypic effect of RNA injected into the worm C.elegans. It was earlier known that antisense RNA as well as sense RNA could silence genes, but the results were inconsistent. They established that annealed sense/antisense RNA, but neither antisense nor sense RNA alone, caused the predicted phenotype. The main results can be summed up as follows:

• Silencing was triggered efficiently by injected dsRNA, but weakly or not at all by sense or antisense ssRNA.

• Silencing was specific for an mRNA homologous to the dsRNA; other mRNAs are not at all affected.

• Double stranded RNA had to correspond to the mature mRNA sequence; neither intron nor promoter sequences triggered a response.

• The targeted mRNA is degraded during the process.

• Only few dsRNA per cell were enough for complete sequencing of the targeted gene.

• dsRNA effect could spread between tissues and even to the progeny, suggesting a transmission of the effect between cells.

Furthermore, RNAi could provide an explanation for the phenomenon studied in Petunias i.e. post transcriptional gene silencing. At the end it was proposed that dsRNA could be used by the organism for physiological gene silencing. Within a year, the presence of RNAi had been documented in many other organisms including Fruit flies, Trypanosomes, Plants, Planarians, Hydra, Zebra fish and humans as well.

'Mechanism of RNA Interference

RNAi is an RNA dependent gene silencing process that takes place in the cytoplasm of the cell. The biochemistry of RNAi was elucidated in an in vitro system based on Drosophila embryo extracts. The hallmark of RNAi is that it is triggered by dsRNA that cause selective gene silencing however, only one of the two strands, which is known as guide strand participate in the process. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of MicroRNA ( miRNA) as well as Short interfering RNA( siRNAs) produced from foreign dsRNA. MicroRNAs are genomically encoded noncoding RNAs that helps regulate gene expression, particularly during development. A miRNA is expressed from a much longer RNA coding gene as a primary transcript known as pre-miRNA, which is processed in the cell nucleus to a 70- nt stem loop structure called a pre-miRNA by the microprocessor complex. The dsRNA portion of this pre-miRNA is bound and cleaved by dicer (an RNase III enzyme that shows specificity for dsRNA and cleave them into uniformly sized small RNAs of 12 to 25 nt ) to produce mature miRNA. Exogenous dsRNA initiates RNAi by activating the dicer, which binds and cleaves dsRNAs to produce double stranded fragments of 20-25 bps, called small interfering RNAs. Small double stranded RNA molecules (miRNAs & siRNAs) can stimulate at least four distinct types of responses that trigger specific gene inactivation: 1. Destruction of mRNA 2. Transcriptional silencing and chromosomal rearrangement. 3. Inhibition of translation 4. Activation of cellular pathways

1. Destruction of mRNA

In animals and plants, the introduction of long dsRNA induces selective mRNA destruction. The long dsRNA are recognized and processed to small pieces of 21-25 nt siRNAs and miRNAs. Rde-1 (RNAi defective) / ago-1 (argonaute) family of proteins and the Dicer, a multidomain RNase- III enzyme mediate these processes. The siRNAs and miRNAs generated by the RNAi process invariably contain two perfectly complementary RNA strands. Guide strand functions to guide the RNA- induced silencing protein complex (RISC) to the target mRNAs and induce their destruction through cleaving the mRNA in the middle of the target region by an as yet unknown nuclease. This guidance of RISC to target mRNA is highly specific to the extent that even 1-2 nt difference in targeting recognition sequence hampers RNAi function.

2. Transcriptional silencing and chromosomal rearrangement

Components of the RNAi are also used for the maintenance of the organization and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serves to down regulate genes pretranscriptionally; this process is referred to as RNA induced transcriptional silencing (RITS), and is carried out by a complex of proteins called RITS complex which contains argonaute, chromo domain protein chp1, and RdRP proteins. RITS forms a complex with siRNA complementary to the local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that are initiated by RNA polymerase. The formation of heterochromatic region requires Dicer to generate the initial component of siRNAs that target subsequent transcripts. Heterochromatin maintenance has been suggested as a self-reinforcing feedback loop, as new siRNA are formed from the occasional nascent transcripts by RdRP for incorporation into local RITS complexes.

3. Inhibition of Translation

In contrast to siRNA, small temporal RNA molecules (stRNA), which represent a large group of small transcripts, called micro RNA (miRNA) mediate gene suppression by inhibiting translation of target mRNA. stRNA are ~70 nt RNA molecules that are predicted to adopt stem lop folds which are further processed to 20-25 nt transcripts. Typically, stRNA recognize the target mRNA by a partial complementary interaction to regions at the 3’untranslated region (UTR) of target mRNA. These interactions direct the inhibition of translation of these genes by an unknown mechanism. Processing of stRNA requires Dicer but rde-1 is not required, alg-1 and alg-2 function to recognize stRNA.

4. Activation of cellular pathways

Long dsRNA can also activates many cellular pathways, some of which can lead to apoptosis. The dsRNA-dependent protein kinase (PKR) is an important antiviral detector protein and is activated by binding to long dsRNA, such as those produced by viruses. Once PKR is activated, it inhibits protein translation by phosphorylating the alpha- subunit of translation initiation factor (TIF alpha).This is followed by a general suppression of protein synthesis that directs the cell towards apoptosis. PKR also activates the 2’5’-oligoA synthetase/ Rnase L enzymes, which are central components of an important antiviral pathway. On binding the dsRNAs, 2’5’-oligoA synthetase is activated, generating oligoadenylates that activate Rnase L, which in turn degrades RNA. DsRNA can also induce interferon (IFN)-alpha and beta expression, which, in conjunction with the other signals, can stimulate apoptosis. Because siRNAs are <30 bp long, they fail to activate the PKR and interferon pathways efficiently.

Evolution of RNA Interference

RNAi is thought to have evolved about a billion years ago, before plants and animals diverged. The process exists in many living organisms, including single celled organisms, plants and human beings. Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway entirely. Most or all of the components of RNAi are also missing in some fungi, most notably in the Saccharomyces cerevisiae. Certain ascomycetes and basidiomycetes are also missing RNAi pathways. It indicates that protein required for RNAi have been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantages in certain niches. Current thinking suggests that RNAi evolved as a cellular defense mechanism against invaders such as RNA viruses, and to combat the spread of genetic elements called transposons within a cell’s DNA.

RNAi – Variation among organisms

Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNAi can be both systemic and heritable in plants and C.elegans, although not in Drosophila and mammals. In plants, RNAi is thought to propagate by the transfer of siRNA between cells through plasmodesmata. The heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of cells. A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNA; In plants, miRNAs are usually perfectly or nearly complementary to their target genes. In animals, miRNAs tend to be more divergent in sequence and induce translational repression.

Biological Functions of RNAi'

RNA Interference has proven to be important for both the development of an organism and the physiological functions of cells and tissues. An intact RNA I machinery is an important requirement for this purpose. Various biological functions of RNAi are as follows

• Immunity against Viral Infections

Antiviral mechanism of RNAi is at work in plants, worms, and flies. The RNAi machinery can recognize invading double stranded viral RNA (or the replicative form of viral RNA) and suppress the infection by degradation of viral RNA. Even before the RNAi pathway was fully understood, it was known that induced gene silencing in plants could spread from stock to scion plants via grafting. It allows the plant to respond to a virus after an initial encounter. Some plant genomes also express endogenous siRNAs in response to infection by specific type of bacteria. These effects may be part of a generalized response to pathogens that down regulate any metabolic process in the host that aid the infection process. The role of RNAi in mammalian innate immunity is poorly understood. It is still unclear how relevant it is for vertebrates, including man.

• Keeping Mobile Elements Silent :

It has been argued that RNA silencing could represent an “ immune defense” of the genome. Close to 50% of our genome consists of viral and transposon elements that have invaded the genome in the course of evolution.It has been proposed that in transposon- containing regions of the genome, both strands are transcribed, dsRNA is formed, and the RNAi process eliminates the undesirable products. As short dsRNA can also operate directly on chromatin and suppress transcription; this would be another mode to keep transposons inactive.

• Down regulation of genes

Endogenously expressed miRNAs, including both intronic and intergenic miRNAs are most important in : Translational repression;

Regulation of development, especially at the timing of morphogenesis; and

Maintenance of undifferentiated or incompletely differentiated cell types such as stem cells.

Thus, miRNA control of gene expression represents a new major principle of gene regulation. Today, it is estimated that there are about 500 miRNAs in mammalian cells, and that about 30% of all the genes are regulated by miRNAs. In plants, the majority of genes regulated by miRNAs are transcription factors; thus miRNAs activity is particularly wide ranging and regulates entire gene network.

• Up regulation of genes

RNA sequences (siRNAs & miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon called dubbed RNA activation. Complete mechanism is still unknown, although dicer and argonaute are involved and there is histone demethylation.

• Crosstalk with RNA editing

Some pre-miRNAs under go A→I RNA editing where adenosine nucleotides are converted into inosine in double stranded RNA via enzyme adenosine deaminase. This mechanism regulates the processing and expression of mature miRNAs. Furthermore, A→I RNA editing may counteract RNAi silencing of endogenous genes and transgenes.

• Evolutionary significance

RNA interference genes, as components of the antiviral innate immune system in many eukaryotes, are involved in an evolutionary arms race with viral genes. Some viruses have evolved mechanisms for suppressing the RNAi response in their host cells, an effect that has been noted particularly fir plant viruses. Studies of evolutionary rates in drosophila have shown that genes in the RNAi pathway are subject to strong directional selection and are among the fastest evolving genes in the drosophila genome.

Technological Applications of RNA Interference

• Gene Knockdown

RNA interference has become acknowledged as an effective and useful tool to study gene function in diverse group of cells. Double stranded RNA is synthesized with sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. There will be a decrease in the expression of targeted gene. By studying the effects of decreased expression, physiological function of the gene can be known. Virtually any gene of the cell can be made the target. Since RNAi may not totally abolish the expression of the gene, this technique is sometimes referred to as “Gene Knockdown” to distinguish it from “Gene Knockout” technique in which expression of a gene is entirely eliminated.

• Functional Genomics

Functional genomics is an attractive technique for genome mapping and genome annotation. Artificial neural networks are frequently used to design siRNAs libraries and to predict their likely efficiency at gene knockdown. Mass genomics screening is widely seen as a promising method for genome annotation and has triggered the development of high throughput screening methods based on micro arrays. It is particularly important technique for genome mapping and annotation in plants because many plants are polyploidy, which presents substantial challenges for traditional genetic engineering methods. RNAi has been successfully used for functional genomics studies in bread wheat (which is hexaploid), Arabidopsis and Maize.

• RNAi Therapeutics

RNAi is a natural process of gene silencing that occurs in organisms ranging from plants to mammals. By harnessing the natural biological process of RNAi occurring in our cells, the creation of a major new class of medicines, known as RNAi Therapeutics, is on the horizon. RNAi therapeutics targets the cause of diseases by potently silencing specific RNAs (mRNAs), thereby preventing disease causing proteins from being made. Among the first applications to reach trials were in the treatment of Macular degeneration and respiratory synctial virus. Other proposed clinical; uses center on antiviral therapies which includes:

Knockdown of host cell receptors and co receptors for HIV;

Silencing of Hepatitis A & B genes;

Silencing of Influenza gene expression etc.

RNA Interference is often seen as a promising way to treat cancer by silencing genes differentially up regulated in tumor cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which involves viral vector systems.

• RNAi Based Therapy

There are some viral infections, the treatment of which is still not possible using traditional methods available. HIV is one such viral infection. Current preventive measures are insufficient to cure the patient because treatment of infected patients results in the emergence of drug resistant virus. RNAi based gene therapy has provided an alternative strategy to block HIV-1 replication. Different types of short and long hairpin RNAs are tested for HIV inhibition in human cells. Primary T cells and later stem cells are transduced with the best inhibiting RNA lentiviral vector system, leading to HIV-1 resistant blood cells.

• RNAi in Biotechnology

RNA Interference has found its place in various aspects of biotechnology. Biotechnology is a vast area where the natural process of RNAi can make a remarkable difference in the already existing methods of improvement and has the potential to open new areas in the field of biotechnology.

Turning non edible plant products into edible products

RNAi has emerged as an important technique to engineer the food plants that produce lower levels of natural plant toxins. E.g- Cotton seeds are rich in dietary proteins but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta- cadinene synthase, a key enzyme in gossypol production, without affecting the enzyme’s production in other parts of plants. Similarly the levels of cyanogenic natural product Linamarin have been reduced in Cassava plants.

Pest control in Livestock

Ticks are distributed worldwide and significantly impact animal health. Due to severe problems associated with the continuous use of acaricides on animals, alternative strategies are being found to manage tick infestation and tick borne diseases in livestock. The use of RNAi provides an efficient global approach for identification of vaccine antigens. Furthermore, it reduces the use of animal challenge experimentation and allows rapid screening of protective tick antigens. Using RNAi, three tick protective antigens; BM86, BM91 and Subolesin were identified in the one host tick, Boophilus microplus.

Disease control in Livestock

With the recent concerns over Prion-mediated diseases (transmissible spongiform encephalopathy) in livestock and their potential transmission to humans, an RNAi based technique is developed for silencing the expression of the Prion protein (PrP) in goats and cattle. Lentiviral based delivery of shRNA targeting the specific gene was effective at reducing the expression of gene. So a stable cell line and a cloned transgenic goat fetus were generated with drastically reduced expression of PrP, the causative agent of neurogenerative disease. Foot and mouth disease virus (FMDV) infection is a contagious vesicular disease affecting various species of cloven hoofed animals, including domestic animals such as cattle, buffalo and goats. RNAi can provide a strategy to control this dreadful disease. Treatment with recombinant replication defective human adenovirus 5 (Ad5) expressing short hairpin RNA directed against either structural protein 1D (Ad5-NT21) or polymerase3D (Ad5-POL3D) totally protects the cells from homologous FMDV infection, whereas it inhibits heterologous FMDV replication. The inhibition is rapid and specific.

CONCLUSION

The discovery that cells have a special mechanism for suppressing the expression of homologous genes by recognizing and processing dsRNA was totally unexpected and has dramatically expanded our knowledge of gene control. The discovery of RNAi has not only provided us with powerful new experimental tools to study the function of genes but also raises expectations about future applications of RNAi in medicine.