Natural GMOs Part 165. Novel double-stranded RNAs get served up in many conventional crops — such as rice.

(An earlier version of this post appeared at GMO Pundit blog.)
Some people have raised worries about whether novel double-stranded RNA molecules present in plant foods might be harmful to people.
Naturally occurring plants often produce novel double-stranded RNA molecules, so we have long been exposed to this potential risk.
One example of a novel plant double-stranded RNA (that was not introduced by lab genetic engineering) has been characterised in detail by Japanese scientists Makoto Kusaba and colleagues (see science reference below). It is an RNA that is present in low protein variety of rice called  LGC-1 that is used as a diet therapy for patients with kidney disease. 
 The LGC acronym stands for Low Glutelin protein Content. Glutelin proteins are a major forms of protein storage inside rice seeds.
These Japanese scientists have analysed the fine detail of the LGC-1 mutation conferring this low-protein production in rice and discovered a mechanism that explains the low protein content of the rice. It is caused by gene silencing triggered by a plant double stranded RNA that gets generated from the mutated region of the chromosome that has been altered by the LGC mutation. 

Chromosomal change causing formation of double-stranded RNA and gene-silencing.
The structure of the region of the chromosome carrying this LGC-1  mutation was shown by the Japanese scientists  to be  substantially reconfigured from the parental, non-mutant version by a presumably accidental spontaneous or natural radiation induced DNA rearrangement that deleted some DNA. 
This DNA deletion of DNA fuses together two very similar genes called GluB4 and GluB5 (that both encode almost identical variants of the seed storage protein glutelin). Most importantly these two genes are close together on the same chromosome, but are oriented in opposite directions — pointing towards one another.
To the right is a diagram from the paper comparing chromosome structures in the region where these GluB genes  reside.
The upper part which is labelled NM describes chromosome of non-mutant (parental) rice. The lower part of the diagram labelled LGC-1 describes the corresponding chromosomal structure in the low protein content mutant rice. The region present in the parental chromosome but missing in the mutated LGC-1 version of the chromosome is encircled in red in the diagram. The directions in which the genes are read by the gene transcription machinery that makes RNA message is shown by arrows.
As a result of the LGC-1 DNA deletion, the mutant chromosome carries a slightly truncated version of gene GluB5 fused to gene GluB4. 
Because the GluB5 gene is oriented in the opposite direction to GlutB4 (see arrows on the diagram), in LGC-1 mutant plants, transcription of RNA starting at the GlutB4 gene and reading through GlutB5 would produce an RNA that is complementary to that normally encoding GlutB5 protein.
The consequence of this natural genetic fusion is that during gene expression that starts at the GluB4 gene in rice cells, the RNA message molecule generated from LGC-1 chromosomal segment form a molecular loop that has a regions of double-stranded RNA corresponding to the inverted highly similar genes GluB4 and GluB5.
In other words, the RNA messages for GlutB4 and the complementary RNA just mentioned for GlutB5 pair up as a double stranded RNA structure analogous to the DNA double helix.
 
As a result of further investigations from Kusuba and colleagues we also understand how the presence of this double-stranded RNA explains the low protein content of this rice.
The low-protein content of  rice grains coming from a crop plant mutant carrying the LGC-1 gene structure arises because of a dominant genetic silencing effect of this double-stranded RNA region on all of the genes of this family of rice glutelin related genes. The double stranded RNA  molecular regions trigger silencing of RNA expression from all the rice genes in this glutelin family. That’s how we get low-protein rice.
This gene family silencing example is also part of a bigger story of the biology of natural RNA silencing, and its relevance to safety assessment of plants containing novel RNA  that will be explored more fully in later posts at this blog. There are several other good examples of natural gene silencing that will be described.
To appreciate the extent of natural RNA silencing, we need to bear in mind that families of multiple duplications of similar genes often occur in plant chromosomes, and analogous DNA arrangements to those that lead to gene fusion in this low protein rice LGC-1 example can easily occur when plant chromosomes of any crop in farmer’s fields are exposed to natural radiation such as cosmic rays.
Studies of crop chromosomes show that multitudinous gene rearrangements and DNA duplications have occurred in nature, so that we can be certain that novel double stranded RNAs and RNA silencing events have frequently occurred during the evolution of crop plants such as rice, maize, soy bean and wheat.
We are thus continually exposed to a natural background novel double-stranded RNA when we eat food, as this might occur when any crop variety has its chromosomes structurally shuffled by DNA damaging natural radiations.
Research Paper:
Low glutelin content 1: A Dominant Mutation That Suppresses the Glutelin Multigene Family via RNA Silencing in Rice
Makoto Kusaba, Kenzo Miyahara, Shuichi Iida, Hiroyuki Fukuoka, Toshiya Takano, Hidenori Sassa, Minoru Nishimura and Takeshi Nishio
The Plant Cell June 2003 vol. 15 no. 6 1455-1467
 
 

Written by David Tribe

David Tribe’s research career in academia and industry has covered molecular genetics, biochemistry, microbial evolution and biotechnology. He has over 60 publications and patents. Dr. Tribe's recent activities focus on agricultural policy and food risk management. He teaches graduate programs in food science and risk management as a Senior Lecturer in the Department of Agriculture and Food Systems, University of Melbourne.

26 comments

  1. Our food safety watchdog, FSANZ, is relying on assumptions instead of seeking evidence when confronted by a newly identified risk in GM foods.
    When FSANZ says it is not “likely” that small dsRNAs in foods will harm humans, it effectively acknowledges this is still possible, and so a risk. Yet it proposes not even testing for that risk until the “weight of evidence” suggests it is doing harm. We say consumer protection should be forward looking – do the tests now: don’t wait for harm to be proven.
    In responding to our peer-reviewed research, FSANZ does not deny that it uses assumptions, rather than scientific testing, to address the risks we identified.
    FSANZ is silent on what specific tests and techniques it uses to guard against unintended effects from the new dsRNA molecules that it is approving as safe for use in GM food crops. What we do know is that it does not require animal feeding studies of any kind. In the approvals that we reviewed, it had never even required tests for detecting dsRNA in the blood of animals, much less required tests that would reliably detect unintended harmful effects from dsRNA. FSANZ does not monitor for effects on people after approval or specify any particular monitoring be done by the developing companies. FSANZ needs to do more than just say its processes work; it needs to be forthcoming on what evidence it relies on to show that all these new dsRNA molecules are no threat to humans.
    To come to its position, FSANZ takes our statements out of context, deflects the issues, and makes misleading assertions.
    The “weight of evidence” it rests its opinion upon is either not appropriate for the testing of new dsRNAs in food, or invokes an absence of evidence when no evidence was ever sought. FSANZ should require this testing as it has the power to in its legislation, it has the option to under international food safety guidelines, and it has a responsibility to the people of Australia and New Zealand to do this.
    Contrary to what FSANZ asserts, there is scientific basis for suggesting that small dsRNAs present in some GM foods may pose a greater risk than those already naturally abundant in conventional foods. Some of these molecules are proven as pesticides. They can have potent effects on animals and should be tested before use on humans.
    RNA molecules are in the food we eat, but to extrapolate from the safe use of food with naturally occurring forms to those that are engineered and unique to new kinds of food is wrong. Proteins of all kinds are also in the food we eat but new proteins are evaluated for the potential to be toxic or allergenic in food. These dsRNA molecules can participate in fundamental biological reactions in human cells and so must be tested to be determined safe.
    Let’s use scientific evidence to see if the new dsRNAs in approved and future foods are safe. Science and public health will then be the winner, whatever the outcome.
    Heinemann, J. A., Agapito-Tenfen, S. Z. and Carman, J. A. (2013). A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environ Int 55, 43-55.

  2. Are you quoting from the article by Jack Heinemann Susan, or is that your text. It’s hard to work out from the way you present it in the comments.

  3. What means novel RNA? Is this new found regulatury RNA ? Or it is artificial RNA? ( I dont want any artificial regulartury RNA in my food! It might regulate me in a form I dont want)

    1. I’m using a term coined by Jack Heinemann. I take it to mean a double stranded RNA that has originated in our food relatively recently, by whatever means.
      Food crops are continually changing due to evolution and breeding. They are not immutable.So you are continually exposed to novelty, whether you know it or not.It is part of biodiversity.
      In breeding new crops or trying new foods there are many opportunities for such novel exposure to natural novel RNAs. These can occur by natural mutations that occurred recently, or mutations from wild crop relatives like grasses that we don’t eat routinely that cross pollinate into our food crops.
      A new food like quinoa to Europeans might present some novel double stranded RNA. But already we have a huge diversity of these RNAs in our food. (in the same vein, we eat lots of novel proteins)

    2. Ute,
      I have added a link to the text near the top of the article that shows the massive amount of variation that occurs when wheat hybrids form. These hybrids between different grasses are called allopolyploids.This occurred in early agriculture, and wheat breeders use them to create new wheats. This breeding triggers lots of changes in wheat chromosomes, and the processes involve variation in levels of ds-RNAs.
      My message is
      http://gmopundit.blogspot.com.au/2013/05/worried-about-ds-rna-worry-about-wheat.html
      Worried about ds-RNA? Worry about wheat and bread.

  4. Seems like the type of argument whose design is to muddy the waters. To me, it sounds like saying, well see, plants in nature mutate in ways we don’t fully understand and affect us in ways we don’t understand, so…therefore GMOs are innocuous because they’re affecting us in ways we don’t understand also.
    Faulty argument because GMOs—and I am NO SCIENTIST—are not merely mutated versions of themselves. They often are spliced with bacteria and other non-plant dna. It seems to my critical and unscientific mind as if a false equivalence is being offered.
    This doesn’t begin to speak of the implications of the increased use of poisons and more roundup that will result from round up ready crops, etc.
    To call these natural gmos is misleading. From the feeling of this site, I’d say that might be the intent.

    1. Jason,
      Compared to conventional herbicides, glyphosate (the active ingredient in RoundUp) is much less toxic. So if the health implications of using those other herbicides are obviously detrimental to your health, then why not go with glyphosate and accept that there might be a very small (but so far undetected) chance of adverse effects. Credible epidemiological studies have been carried out (see for example http://www.ncbi.nlm.nih.gov/pubmed/22683395 and http://www.ncbi.nlm.nih.gov/pubmed/21798302) that have shown no significant adverse health effects of glyphosate use. Also, there are crop varieties that are naturally glyphosate-resistant (and can probably be grown organically without the use of glyphosate spraying) but no-one bangs on about them.
      Second, gene transfer between the kingdoms of life does occur in nature (see for example this freely available article at http://www.plantcell.org/content/21/7/1897.full). It is rarer in plants and animals simply because (1) the physical gene DNA has a harder time to get into the acceptor cell unharmed and insert itself into the genome, (2) the gene must confer some kind of selective advantage to the plant – otherwise it will quickly be lost or inactivated by random mutations, and (3) because the different kingdoms of life differ in gene structure (exon-intron borders, transcription initiation and termination sites etc), codon frequencies and so on. So just because a “foreign” gene naturally makes it into a plant genome unharmed and could possibly be very useful to the plant, the plant might not be able to successfully express the gene and it will be lost. Genetic engineering is simply taking all three aspects into consideration when modifying a crop plant. Imagine a scenario where glyphosate for some reason was abundant in nature (and not sprayed by us) and if the target gene in plants (the enzyme 5-enolpyruvylshikimate-3-phosphate synthase) could not become naturally resistant – then chances are that the same gene transfer event would occur “naturally” i.e. a bacterial version of 5-enolpyruvylshikimate-3-phosphate synthase would integrate into the plant genome.

  5. Jason
    Your version of the argument is not one I agree with. But I do argue for comparative risk assessment of GE crops to identify any new risks not present in the existing version food, and to do this you need to understand what the existing risks are. This is not false equivalence, as equivalence is not assumed or implied – it’s called comparing levels of risks.
    It makes no sense to guard against risks which we ignore as trivial in other food examples — as this actually prevents us making safety and human welfare improvements.
    Natural GMOs include precisely the changes that you, Jason, mention with Gmos, namely bacterial DNA in plants and non-plant DNA. Transgenes often get created naturally.

  6. Hi David. Thanks for the patient clarifications. I clearly stand corrected on multiple facets of the argument I was putting forth. I am outmatched on this discussion due to my lack of genetic knowledge. I will point out, however, that I’m pretty good at logic. As such, I find your argument for using roundup because it is less toxic to be faulty. There certainly are studies that contradict your assertions of safety. As well, it’s ‘less toxic’ status does not make it safe to use, especially since it will now be and is now used in drastically greater quantities. Superweeds and a host of issues come up that I won’t go into.
    This issue is as big and thorny as evolution v creation (which I would add is a false dichotomy) I agree with your first point that, as a baseline, we should test the effects of these natural gmos. However, no-one bangs on about the naturally glyphosate resistant crops because no one is planning on using tons and tons more roundup on these crops. If there were long-term studies on the effects of Roundup use on spray techs and farmers for 30 years or so I might feel better.
    As well, and I’ll speak for those of us who want gmos labeled and who are against the proliferation with fewer and fewer tests by the US EPA, FDA, USDA, etc, by saying that we’re not afraid of technology. We are surrounded and inundated by it for better and for worse. It’s the undue influence, lobbying, misleading, and big-brother gestapo tactics of companies like Monsanto that are alarming.
    Another argument of yours seems odd. You state more clearly than I, that since gmos happen in nature we should study them too for safety. Agreed. However, to draw a comparison between certain plants randomly mutating in nature to intentionally mutating every plant of a crop is a false equivalence. Then this intentionally manipulated plant pollinates its neighbors and they become like the pollinator and there is monoculture as an end-result. These are vastly different end-results.

  7. “This issue is as big and thorny as evolution v creation”
    This isn’t actually an issue, just so we’re clear, hasn’t been since pre 1900…
    if you’re saying what I think you’re saying, regarding evolution and creation, then not only are you lacking “genetic knowledge” but also any capacity to be taken seriously in the arena of scientific knowledge whatsoever.

      1. Which attack?
        There is no attack there. If someone believes, as a reading of Jason’s post suggests, that the evolution vs creation issue is big or thorny (in terms of being an actual debate) then it is simply a statement of fact that they have no capacity to be taken seriously in the arena of scientific knowledge.
        I do, however, enjoy your abject lack of respect for the rules – rules lawyering tends to be frowned upon also.
        Alas now you appear to have actually taken me off topic, but I think that’s one point to you in the realms of breaking the rules rather than me.

          1. Your question doesn’t follow at all from speaking of rules. I’m not entirely sure I follow – I’ve made no claims that there is a specific list, only that a belief that the creation/evolution issue is thorny, unresolved, or an actual (meaningful, as in one in which the evidence isn’t in, the facts aren’t known and we’re down to simply filling out our knowledge and refining our theory) debate is quite clearly a distinctive flag that one has very little likelihood of being remotely correct unless by utter accident when discussing science.

          2. Hey everyone. Thanks for chiming in. David, again thanks for the info. And you’re right: I didn’t cite and I should and will in the future. I still think that based on what I’ve seen and read that using more herbicides and pesticides is a bad idea and I’ll leave it at that.
            Ewan, buddy. You got me. I didn’t write in and correct/clarify myself because I was having too much fun watching you argue with yourself. 🙂 I’m sure I’ll get it for that!
            We can all agree that there is no scientific evidence that casts doubt on evolution. My point was that there was a greater social, cultural debate about the nature of our existence. Sadly, it is cast as evolution v creation. My point was that despite the disagreement, both are essentially true from my perspective. Science, logic, experimentation have been extremely useful but can’t tell us how or why we’re here. The linear has its limits. It seems sad that science can try to disprove God. Where some fundamentalists and others can try to disprove evolution with pseudo-science. Also sad.
            Best wishes all.

  8. Dear Jason,
    Its pleasant conversing with you because you are a fair player in this conversation.
    Unfortunately logic alone is not sufficient to work through well founded judgements on these issues. Factual accuracy, avoidance of bias, and full evaluation of the context are needed before reaching conclusions. In the absence of this, apparently impeccable logic give you the wrong inferences.
    You make claims that genetically modified crops increase herbicide and pesticide exposure, but there is abundant evidence that they allow farmers to switch from using more toxic to less toxic chemicals. If they had not made that switch the environment and farmers would have had far greater exposures of toxic chemicals. That is the comparison to make. It’s called the counterfactual comparison.
    You beg to differ with me on the relative environmental safety of glyphosate saying vaguely that there are some publications to the contrary but don’t document them. There is however a substantial body of evidence that the environmental impact of glyphosate is low compared to other herbicides used. It’s not just the few minority studies that prove the point, is the total amount of information and the relative quality of all the different studies that should be used in making a conclusion. One swallow doesn’t make a summer.
    I would argue that your “false equivalence” argument, amounting to saying that we never get exposed to large quantities of produce from a natural mutant is a good example of where your lack of understanding of the context and your lack of factual accuracy about how new crop germplasm is used in conventional, non-GM breeding leads you to make false inferences.
    The nature of the way in which non-GM (and GM) crops are produced is to do some breeding to introduce novel non-GM crop germplasm into a novel high producing variety, and then bulk up seed from that particular variety for use in farms. This seed multiplication produces large numbers of seed copies containing a limited number of genetic types. Novel mutations introduced at the start of this process are distributed in huge quantities of copies to many fields and can give large-scale human exposure to particular novel but naturally arising genetic types — just as GM does. Evaluation of mutations involves thinking about how they originate, and also thinking about how they multiply in natural systems.
    Some examples of where the low relative toxicity of glyphosate is documented are:
    Disputation of Mn chelation claims about glyphosate:
    http://www.weeds.iastate.edu/mgmt/2010/glymn.pdf
    DEVELOPMENTAL AND REPRODUCTIVE OUTCOMES IN HUMANS AND ANIMALS
    AFTER GLYPHOSATE EXPOSURE: A CRITICAL ANALYSIS Journal of Toxicology and Environmental Health, Part B, 15:39-96, 2012 DOI: 10.1080/10937404.2012.632361
    Amy Lavin Williams, Rebecca E. Watson, John M. DeSesso Exponent, Inc, Alexandria, Virginia, USA
    Glyphosate is the active ingredient of several widely used herbicide formulations. Glyphosate targets the shikimate metabolic pathway, which is found in plants but not in animals. Despite the relative safety of glyphosate, various adverse developmental and reproductive problems have been alleged as a result of exposure in humans and animals. To assess the developmental and reproductive safety of glyphosate, an analysis of the available literature was conducted.
    Epidemiological and animal reports, as well as studies on mechanisms of action related topossible developmental and reproductive effects of glyphosate, were reviewed. An evaluation of this database found no consistent effects of glyphosate exposure on reproductive health or the developing offspring. Furthermore, no plausible mechanisms of action for such effects were elucidated. Although toxicity was observed in studies that used glyphosate-based formulations, the data strongly suggest that such effects were due to surfactants present in the formulations and not the direct result of glyphosate exposure. To estimate potential human exposure concentrations to glyphosate as a result of working directly with the herbicide, available biomonitoring data were examined. These data demonstrated extremely low human exposures as a result of normal application practices. Furthermore, the estimated exposure concentrations in humans are >500-fold less than the oral reference dose for glyphosate of 2 mg/kg/d set by the U.S. Environmental Protection Agency (U.S. EPA 1993). In conclusion, the available literature shows no solid evidence linking glyphosate exposure to adverse developmental or reproductive effects at environmentally realistic exposure concentrations.
    Williams, G. M., Kroes, R., and Munro, I. C. 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Reg. Toxicol. Pharmacol. 31: 117-65.
    Crossan and Kennedy, isnapshot of herbicide tolerant cotton.
    https://docs.google.com/file/d/0B7hhP5QasNtsaFN6NU0tZmYydXM/edit?usp=sharing

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