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Home > Our Products > Technical & Safety Info > For the Record - Science > RNA Interference in Plants

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RNA Interference in Plants

Introduction - The Basics of Life

The genetic information for all living things is found in DNA (deoxy-ribonucleic acid). This genetic material encodes the information that ultimately shapes what an organism is, controls how it grows and develops, and influences how it behaves. Selective activation and inactivation of genes encoded in DNA enables an organism to produce specific proteins (long chains of amino acids) in particular amounts over time and in response to the internal and external environment of the organism.

The production of protein takes place in two steps. In the first step, called transcription, the permanent DNA message (Figure-1, 1) is copied into a temporary messenger RNA (mRNA, 2) by an enzyme (RNA polymerase). This mRNA message can be read by a complex cellular “machine” called a ribosome (3).  In this second step, called “translation,” the ribosome assembles amino acids in an order specified by the mRNA to create a specific protein (4).

The growth, development, and function of an organism is a reflection of gene expression- the timing, pattern, and quantity of mRNA and the production of proteins from genes within the organism.  Thus, gene regulation is essential to life- from the simplest virus to the most complex mammal.

Gene expression and protein function are controlled at all levels. DNA activation, mRNA production and degradation, and protein production, activity, and degradation are controlled by a variety of signals from inside and outside the cell. Much of gene regulation is mediated by proteins, but in the past decade scientists have discovered that gene regulation is also mediated through gene silencing induced by short segments of double-stranded RNA (dsRNA). Several regulatory mechanisms are known, and will be referred to collectively as RNA Interference, or RNAi.

History of RNAi

The story of RNAi begins with unexplained genetic phenomena observed in plants in the early 1990s by Jorgensen (white petunias) and by Baulcombe (viral resistance), and later observed in animals. The discovery that dsRNA was the agent responsible for these findings was published in the journal Science in 1998.  The 2006 Nobel Prize was awarded to Fire and Mello for this discovery. RNAi is already widely used to study gene function and has led to novel medical applications. RNAi was used commercially in tomatoes as early as 1994, although the mechanism at that time was referred to as “anti-sense suppression.” Monsanto continues to investigate the use of RNAi to improve plants used for food and feed.

How Does RNAi Work?

Most RNA is single stranded and is produced by transcription as described above.  RNA can also form double-stranded RNA (dsRNA) if it carries a sequence of base pairs that allows it to fold back and match up with itself.  dsRNA is similar to the famous “double helix” of DNA, formed by zipping together matching base pairs on the complementary strands of DNA.

RNAi begins (Fig. 2, 1) with a dsRNA within a cell. This dsRNA segment may be normally occurring within the cell, may be added to the cell through genetic engineering, or may be present due to a viral infection.

Small dsRNA molecules are produced within a cell when a longer dsRNA is processed in the nucleus by an enzyme (protein) called “dicer” (2), which cuts the dsRNA randomly into sections 18-21 base pairs in length.  These short segments of dsRNA (3) then participate in one or more of several gene regulation pathways described below.

The first of these pathways (4), involves the RNA-Induced Silencing Complex (RISC), or “slicer”, which uses short segments of dsRNA to identify and bind to a matching mRNA.  The mRNA is then cleaved, preventing protein production. The resulting fragments of RNA are recycled by the cell.  This gene regulatory process appears to be particularly important in plants.

Another pathway (5) uses the short dsRNA to bind to mRNA without cleaving the molecule.  Binding typically takes place near the end of the message, and shuts down translation of the mRNA to protein. Characterization of the precise mechanism is ongoing, but this pathway appears to be more important in animals.

Finally (6), the short dsRNA can apparently bind to DNA, controlling the transcription of DNA to messenger RNA and down-regulating gene function. Again, the precise mechanisms and enzymes involved in this process are still being investigated. This can occur in plants and animals, and the overall importance of this pathway is not fully characterized.

Bio-Medical Importance of RNAi

RNAi is an important tool in medical research because it can reliably suppress selected genes, allowing scientists to determine the function of a gene. That knowledge can be used to develop new diagnostic and therapeutic tools. It is now clear that RNAi plays an important role in human gene regulation, but scientists are only beginning to understand the importance of this discovery.

Medical treatment using RNAi is in its infancy, but has already resulted in a promising therapy for one type of blindness (macular degen-eration). RNAi shows great promise for treating cancer, and it may be possible to use RNAi to target cancer cells while sparing normal tissue. Other areas of promise include allergic and inflammatory diseases such as arthritis, viral infections (hepatitis, HIV/AIDS), elevated cholesterol and other lipid disorders, and inherited disorders such as Lou Gehrig’s disease (ALS).

The great challenge for RNAi therapy is delivery. It is very clear that ingestion or intravenous injection of dsRNA does not work in mammals, probably due to rapid breakdown of RNA and the lack of an uptake mechanism to move RNA into cells. Thus, RNAi in humans must use local non-systemic delivery routes (e.g. intraocular injection) , chemically modified (stabilized) RNA, stable RNA “look-alikes;” high doses, lipid delivery agents, or gene therapy to introduce new DNA that can make dsRNA inside the cell.

While these limitations make delivering RNAi-based therapy more difficult, they also indicate that exposure to dsRNA in food or feed will produce no effect in mammals.

Using RNAi in Genetically Modified Plants

A source of dsRNA must be introduced into a plant’s DNA to create plants that use RNAi-mediated traits and that pass those traits on to the next generation.  This is done using the same techniques of genetic modification used to produce other biotechnology plants being grown today. The RNAi-mediated trait can be introduced into the plant genome as a DNA molecule made from opposite strands of the gene to be suppressed.  One segment is inverted with respect to the other, and the segments are separated by a short “loop” segment (Figure-3) so that the resulting RNA can fold back on itself to form a double stranded structure. In practice, segments of more than one gene can be included to provide suppression of multiple genes.

Applications: Healthier Oils

Consumers are always searching for foods that provide health benefits. Monsanto is exploring RNAi to maximize the production of beneficial mono-unsaturated fats in soybean, while minimizing saturated fats and eliminating trans-fats. This technology can also reduce polyunsaturated fats which, although beneficial, are unstable and make oils unsuitable for some applications. Thus these improved oils are not only beneficial, but can also be used in a wider variety of foods without sacrificing food flavor or quality.

Applications: Pest Control

The digestive system in an insect or a nematode is very different from the digestive system in mammals, and we now know that dsRNA designed to suppress specific genes in some pests, can be provided in the diet to suppress or kill those pests.  The sequence specificity of RNAi presents the opportunity to selectively target some pest species while sparing desirable species.  Unlike some chemical pesticides, RNAi in plants is not expected to have any effect on non-target insects and nematodes, birds, reptiles, fish, or mammals (see below).

All transgenic-plant-mediated insect control technology commercially available today is based on the plants producing proteins derived from a specific type of bacteria, Bacillus thuringiensis, or Bt. Insect resistance to Bt has not been a significant problem to date, but risk of development of resistance to these proteins can be further reduced with the use of non-Bt-crop or natural refuges of non-resistant plant hosts.  Combining Bt technology with a second, independent mode of insect control via RNAi would both enhance product performance and further guard against the development of resistance to Bt proteins.

RNAi: Safety and Health in Humans And Animals

RNA, and the phenomenon of RNAi, are universal in plants and animals. Recent studies (Ivashuta et al., 2009) confirm that rice contains a very large number of short dsRNA, many of which have sequences matching segments of various human genes.  Thus, we have a long history of safe exposure to dsRNA's that can mediate RNAi in foods, including plant-derived RNAs that match our own genes.  RNA in general is recognized by the U.S. Food and Drug Administration (FDA) as “Generally Regarded as Safe” (GRAS) and as such, consumption is not regulated.

We also know that there are important differences between mammals (including humans) and many lower organisms such as plants, insects, and nematodes. Lower organisms appear to perpetuate the RNAi signal  and distribute the signal cell-to-cell throughout the entire organism. While RNAi does work in mammalian cells, signals in mammals appear to be short-lived and are not known to be transmitted from cell to cell.  Pharmaceutical research has shown that oral dsRNA is ineffective at inducing gene suppression in humans, even when directed at human genes, necessitating highly specialized delivery mechanisms to bypass natural barriers to dsRNA uptake (Akhtar, 2009).

Finally, RNA is unstable in the environment, and is unlikely to survive cooking and other food preparation. In addition, the digestive tract provides significant defenses, as most RNA will not survive the environment of the stomach and intestines where secreted enzymes and the normal bacterial flora of the gut will likely degrade RNA. In fact, development of delivery systems that can overcome the natural barriers preventing the oral use of RNA is a very active area of pharmaceutical research.

In order to exert systemic effects, RNA would need to overcome the barriers of the digestive system, survive circulation in the blood stream, and transit the membrane barriers and cytoplasm of the cell in order to reach a target for gene suppression (mRNA).  Since attempts at gene suppression using oral dsRNA in mammals have met with significant challenges, the possibility of RNA in foods resulting in adverse effects in humans appears to be exceedingly remote. If gene-specific experiments become necessary to assure the safety of products based on RNAi, they will be conducted as part of the food safety assessment.

RNAi: The Environment

The environmental safety assessment approach to RNAi is still under development. RNAi and other forms of RNA-mediated gene regulation are ubiquitous in the environment because they are important in all living things. Despite this, we are not aware of environmental interactions mediated by double-stranded RNA.  The stability of free RNA in the environment is limited, and as discussed above, mammals as well as birds and reptiles, effectively resist the effects of exogenous RNA.

For non-pesticidal uses of RNAi, there is little reason to expect effects beyond the modified plant itself.  The need, if any, for environmental studies in this situation remains to be defined.

For pesticidal RNAi, as for any pesticide, appropriate non-target-species and environ-mental fate studies will be performed.  General studies demonstrating a lack of RNAi effect in certain species or classes of animals (such as the lack of effects on mammals) or a lack of stability in the environment may address many issues without the need for gene- and target-species-specific studies. RNAi gene-specific studies on selected, agronomically important organisms may be necessary to address effects on non-target species such as nematodes or insects which are not inherently resistant to RNAi mediated by exogenous RNA.