Scientists Get Research Tips From Cells: How RNAi Became a Cutting Edge Tool in Modern Genetics

Genetics is the backbone of modern biological research. Sequencing genomes has provided insight into evolu- tionary conservation and divergence between entire organisms down to the level of single domains within proteins. Proteins are the functional output from gene sequences, and are the structural support and functional units of cells, controlling events such as growth, differentiation, movement and proliferation. Understanding the structure and function of each protein provides insight into the intricate molecular framework of all cells and their components. Sequencing has also allowed the cellular process RNA interference (RNAi) to be identified and applied, as will be discussed in this article, as a genetic and a therapeutic tool for the manipulation of protein expression. 

 If you imagine a cell as a warehouse, then genes, encoded by DNA, are the supervisors, RNA are the secretaries, small factors such as Ca2+  are the tea room biscuits and proteins would be the scaffolding, the machinery and the workers. Considering their importance in the cell, scientists have gone to great lengths to expose the structure and function of pro- teins so they can uncover all of the signaling pathways and interactions that proteins are involved in. Understanding protein function is especially important in medical research, where the primary aim is to reveal what’s going wrong in a biological system and hence which proteins should be targeted with medication. 

  But when the entire human genome was sequenced, scientists discovered that there are 30,000 to 40,000 genes, all encoding one or more proteins. That’s like a warehouse full of workers that all need to be identified and the supervi- sors don’t even know their names, let alone what they’re all meant to be doing. Where do you begin? Initially, the concept of forward genetics [1] was employed to identify the gene that corresponds to a particular protein. This is done by scrutinizing the effect a mutation has on an organism, and then trying to work out which gene the mutation is in. For instance, there is a factory worker who sleeps on the job and you have to go and find his supervisor Sequencing genomes has provided insight into evolu- tionary conservation and divergence between entire organisms down to the level of single domains within proteins. Proteins are the functional output from gene sequences, and are the structural support and functional units of cells, controlling events such as growth, differentiation, movement and proliferation. Understanding the structure and function of each protein provides insight into the intricate molecular framework of all cells and their components. Sequencing has also allowed the cellular process RNA interference (RNAi) to be identified and applied, as will be discussed in this article, as a genetic and a therapeutic tool for the manipulation of protein expression. 

 If you imagine a cell as a warehouse, then genes, encoded by DNA, are the supervisors, RNA are the secretaries, small factors such as Ca2+  are the tea room biscuits and proteins would be the scaffolding, the machinery and the workers. Considering their importance in the cell, scientists have gone to great lengths to expose the structure and function of pro- teins so they can uncover all of the signaling pathways and interactions that proteins are involved in. Understanding protein function is especially important in medical research, where the primary aim is to reveal what’s going wrong in a biological system and hence which proteins should be targeted with medication. 

  But when the entire human genome was sequenced, scientists discovered that there are 30,000 to 40,000 genes, all encoding one or more proteins. That’s like a warehouse full of workers that all need to be identified and the supervi- sors don’t even know their names, let alone what they’re all meant to be doing. Where do you begin? Initially, the concept of forward genetics [1] was employed to identify the gene that corresponds to a particular protein. This is done by scrutinizing the effect a mutation has on an organism, and then trying to work out which gene the mutation is in. For instance, there is a factory worker who sleeps on the job and you have to go and find his supervisor because he is slowing down production. It doesn’t sound very forward, but the use of this concept has been instrumental in the characterization of many genes and their corresponding proteins. 

  Now that sequencing is easy and relatively cheap, however, there is a new way to determine the gene to protein correlation. It is the process of reverse genetics [1,2], and it involves first knocking out, that is, removing the function of, a gene of interest and then observing the pathways and processes that have been disrupted because of the loss of the protein encoded by that particular gene. This process would be just like kidnapping that supervisor and seeing all of the trouble the workers get into. Reverse genetics is ironically straight forward, while forward genetics is a little reverse. As in every industry, there is always a new process that makes the job easier. RNAi is an exciting tool that can be used to advance reverse genetics, though it was not designed by anyone for that purpose. Andrew Fire and Craig Mello discovered RNAi in the worm Caenorhabditis elegans, noting that when double stranded RNA (dsRNA) is injected into the organism, the complementary messenger RNA (mRNA) gets degraded and the protein is not produced [3]. One final warehouse metaphor before we get into the nitty-gritty: each worker (protein) has one secretary (mRNA) who has come from the supervisor (gene). When a secretary is kidnapped (mRNA is degraded through RNAi), the worker doesn’t come in to work (protein is not translated) and so we can observe what happens in the warehouse without that worker (reverse genetics). That is the main concept of RNAi.  RNAi is indeed more complex than this simple notion. There are several classes of double stranded RNA (dsRNA) that can elicit RNAi; short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) and micro RNAs (miRNAs). Each class has a different and complex role in the cell (reviewed in [4]). One function of RNAi is to protect the cell from long dsRNAs, such as those introduced by viruses. Viral RNA employs host cellular machinery to create viral proteins in the cytoplasm that are used to replicate the virus. Generally, viruses damage the host cell while it’s infected and will destroy the cell when leaving it, giving the cell a pretty good reason to destroy the virus first. RNAi machinery will identify and target the dsRNA in the cytoplasm and cleave it into a certain size [5]. These strands, or siRNAs, are incorporated into another complex [6] that targets any complimentary mRNA that has been developed, and cleaves that also [4,7]. Overall this system is fairly effective, as it also stops viral proteins being made. As we know, however, viral infections still occur, so RNAi doesn’t always make it in time. 

  The other main role of RNAi is to manage the cells own gene transcription thereby controlling the amount of protein that is produced from an mRNA [4]. This occurs when dsRNAs are produced from the genome of the cell, which are then cleaved into smaller fragments, or miRNAs, and incorporated into the same complex as in the above pathway. When there is a perfect match between the mRNA and the ‘guide strand’ of the miRNA, mRNA is cleaved. If the mRNA is less complementary to the miRNA, the mRNA is destabilized, causing it to be degraded faster than other mRNAs so that less protein will ultimately be made from it. But how have researchers used this technique to knock out proteins that they’re specifically interested in? They have designed a way to hijack the cellular RNAi machinery, but not disrupt the normal process [8]. This involves using transgenic techniques to insert shRNA transcripts into the genome of the cell [9], which is produced and processed in a similar fashion to the miRNAs. Because they have been designed to target a specific mRNA, the guide strand is a perfect match and the mRNA gets cleaved, thereby drasti- cally reducing the amount of target protein synthesized [7].  And so, for the past decade, researchers have been utilizing the process of RNAi to remove proteins and observe how the cell reacts. The best way to observe the effects of a knockout phenotype is to study them in organisms such as mice, observing the effects in a physiological context to that similar of a human. Many genetic approaches can be used to do this, including of course RNAi. Few of these techniques can also be made to be even more specific by just deleting a protein in a particular cell type. For example, RNAi is capable of knocking down the expression of a protein in only white blood cells and observing the diseases it causes, for instance leukemia, without disrupting the function of the protein in the rest of the body [10]. This ability is fun- damentally important to research, as it allows researchers to pinpoint exactly what causes a particular disease and hence underpins the development of cures. 

  RNAi can go one better than other techniques, it can be ‘switched on’ in grown mice [11]. Designed shRNAs can be induced when a particular compound is present, so you can feed an adult mouse this compound in water and it will promote the transcription of the shRNA, which will then repress the protein. This has been a major breakthrough in reverse genetics, as a lot of proteins are essential to em- bryogenesis, and those mice that have the protein knocked out die before their phenotype can even be studied. This is also very practical, because it means you can see how use- ful targeting a particular protein will be in an established disease model. For instance, in a cancer model, you can turn on the knockdown of the specific protein to determine if it will really make the tumor regress or even go away. If it works, you know exactly what to target for treatment. RNAi is looking pretty good so far. It’s a natural pro- cess, researchers can harness it for knockdown of specific proteins and it can be ‘switched on’ at any time during the development of an organism. No wonder Andrew Fire and Craig Mello were awarded the Nobel Prize in 2006 for their discovery. But can RNAi really do it all? Recent research has been investigating the potential of using dsRNA to harness the process of RNAi as an actual drug to target human viral infections and diseases [2]. RNAi can be designed to target any protein specifically, so the therapeutic potential is more extensive than that of current medication [12]. There are, of course, hurdles for the therapeutic use of RNAi and it the delivery of dsRNA into human cells. This process can create a immune response [13] via interferons, an integral cellular immune component that recognizes dsRNA 30 nucleotides or longer, as a viral defense mechanism. Another issue is to make sure the cellular RNAi machinery can cope with the amount of introduced dsRNA [12]. 

  Current aims in research today are to design effective and non-toxic delivery methods to initiate therapeutic RNAi in human cells [14]. There are two methods that can be usedto elicit an RNAi response in living animals. Long term RNAi expression involves infection of cells with a virus that will naturally integrate its DNA into the genome of the host cell. This DNA can be engineered to contain the sequence of the shRNA and a promoter so it is expressed in the cell type desired. When the cell divides, all daughter cells will also have the shRNA, thus ingraining the construct in the genetic make-up of the organism. 

 Using viruses isn’t a desired method at the moment as integration is random into the host genome, leaving the possibility that it could insert into and disrupt important genes. Transient infection methods are therefore the current focus of therapeutics [1]. Transient infection involves deliv- ering siRNAs directly to the cell. The siRNA then binds to the RNAi machinery and targets mRNAs, eliciting a potent and predictable knockdown of target mRNA at low doses [2] In this method, the saturation of host RNAi machinery is less likely, though interferon responses are still possible. Transient infections last from 3 to 7 days in rapidly dividing cells and can last weeks in non-dividing cells until siRNAs are degraded [12]. 

 An example of current RNAi research is aimed against HIV (Human Immunodeficiency Virus) infection [15]. Our genome does in fact provide us with some protection against HIV. We produce miRNAs that target two HIV genes that are required for viral replication [2]. However, viral genomes mutate and adapt rapidly, and the HIV genome evolved a gene to suppress the action of those miRNAs. Initial re- search aimed to target the viral genomic RNA and the early transcripts produced within the cell [16], however due to the rapidly evolving nature of the HIV genome, a common trait of viruses in general, miRNAs that were once specific for HIV genes are no longer effective. This is why new ap- proaches are targeting a human co-receptor, called CCR5, which HIV binds to in order to enter our cells. This recep- tor is one of the only receptors that HIV binds that can be removed without harming the cell [15]. Specific CCR5 siRNA could be administered to people in high-risk areas for HIV infection, or soon after a suspected infection, so the virus doesn’t get a chance to infect more cells. This is just one of many examples of viral infection that can be specifically fought using RNAi [17]. 

 For a simple process, RNAi has been applied as a ver- satile and instrumental tool in biological research. In reverse genetics RNAi can be used as a specific means to knockdown the expression of single proteins. In mouse models, RNAi can be switched on to identify which protein targets should be inhibited to make a disease phenotype regress. Now, tools to exploit the process of RNAi are being developed to inhibit viral function and hence protect cells against viral infections. RNAi has the potential to expand its horizons therapeutically with further research in long lasting RNAi expression in human cells. Thus, the potential for RNAi to revolutionize the way we fight infection is truly phenomenal [2,17-19]. It is overwhelming to think that a mechanism to answer the plethora of scientific questions could be found in our cells, and they’re already doing it themselves. If a cell is like a ware- house, then RNA interference is organized crime, kidnapping supervisors at the instruction of the director and external parties, with incredible potential to do significantly more.   

 Sarah Best is an undergraduate at Yale University