Based on invited presentation at 1st Forum of
Development and Environmental Safety, under the theme “Food Safety and
Sustainable Agriculture 2014”, 25 - 26 July 2014, Beijing
Genetic determinism has
long dominated scientific thought, education and public understanding of
evolution and biology in the West, misguiding philosophy, medicine, politics,
public policies, and society at large.
The central dogma of molecular biology describes the linear
flow of genetic information from DNA (deoxyribose nucleic acid) to RNA
(ribosenucleic acid) to protein, with each protein performing a functional role
in the organism . The concept rules out the effects of the environment on
the organisms’ function and heredity. It has provided motivation and
justification for biotechnologies such as genetically modified crops, where the
thinking is that insertion of one gene into a crop or
animal will not have consequences for the rest of the genome, or the organism
as a whole and those exposed to it.
Paradigm shift away from the central
dogma to the fluid genome
Over the last few decades there has been accumulating evidence that the central dogma of molecular
biology is outdated and overly simplified. There is a paradigm shift occurring
in our understanding of our intricate and complex relationship with the
environment, supported by mounting work in the field of epigenetics. A few out
of many examples include work showing that the in utero environment can
influence the development of off-spring as well as their heath prospects long
into adulthood, including life-expectancy and stress-related illnesses , while
malnutrition of either fathers at time of conception of mothers at certain
points during pregnancy, can affect metabolism of grandchildren [3-5]. Learned
behavioural traits are found to pass down the generations [6, 7]; and chemicals
can have transgenerational effects [8, 9].
There are many
players involved in the complex intercommunication necessary for organism
survival that can have transgenerational effects, including the natural genetic
engineering of the genome itself in response to environmental cues (see  Evolution
by Natural Genetic Engineering, SiS 63). Organisms themselves are
able to swap DNA with unrelated species of organisms
in what is now acknowledged as horizontal gene transfer. We know that
the levels to which genes are switched on and off through chromatin based
mechanisms, something arguably as important, or more important than the genetic
sequence itself, are often passed down the generations. Non-coding RNAs have
also been revealed as major players in epigenetic regulation of gene expression
that can also be inherited (see  Artificial
versus Natural Genetic Modification and Perils of GMOs and  Ban GMOs Now, special ISIS for further
details). I shall concentrate on epigenetic effects due to RNA interference.
RNA-mediated epigenetic inheritance and
There are now around 50 known classes of
RNAs most of which are non- protein-coding . Non-coding
RNAs are transcribed from vast regions of the genome dubbed ‘junk’ DNA not so
long ago, as less than 2 % of the human genome codes for proteins. Non-coding
RNAs are strongly conserved between vertebrates, invertebrates and
plants, and appear to be involved in most biological and metabolic processes
from editing DNA and RNA, acting as enzymes, and controlling gene expression.
One mechanism of
RNA-mediated epigenetics is the RNA interference (RNAi). RNAi is a natural
epigenetic process highly conserved in plants, vertebrates as well as
invertebrates in which short non-coding
double-stranded RNAs (dsRNAs) regulate gene expression in a sequence-specific
manner, normally to down-regulate individual genes, or set of genes. Short dsRNAs
includes siRNA (short-inhibitory RNA), miRNA (microRNA), shRNA (short hairpin
RNA) etc., are all intermediates leading to RNA
interference of protein synthesis. The underlying mechanism of RNAi gene
regulation relies on the sequence complementarity of the small RNA molecules to
its target mRNA of a given gene, resulting either in degradation of the target
RNA, or translational repression of the protein product which is often the case
when there is incomplete complementarity to the target sequence. Typically,
dsRNA originates from a long RNA molecule with stretches of complementary base
sequences that base pair to form a stem ending in a non-base-paired loop. This
stem-loop structure is then processed into a shorter dsRNA, and one strand, the
guide strand does the job of interfering. It binds to an mRNA (messenger RNA)
molecule in the cytoplasm by complementary base-pairing to prevent the mRNA
from being translated into protein. Alternatively, the guide strand targets and
chemically modifies DNA sequences in the nucleus by adding methyl groups to the
DNA, and cause modification of histone proteins associated with the DNA (see
 New GM
Nightmares with RNA, SiS 58). The nuclear pathway is known to
inhibit transcription and to seed the formation of heterochromatin, an
inactive, non-transcribed region of chromosomes.
Short dsRNAs can target and regulate hundreds or even thousands of genes, with synthetics
RNAs having an estimated 10 % error rate ,
despite being designed to target specific genes.
Examples of RNAi mediated epigenetics
Two interesting examples of RNAi-mediated
epigenetic inheritance include the Kit paramutated mouse, which was the
first mouse model of RNA-mediated inheritance. Paramutation is the transfer of
an epigenetic state from one allele to another of the same locus such that
heterozygous mutants for the Kit paramutation
can transfer its epigenetic state to the wild-type locus, which is then passed
down to wild-type offspring. Homozygosity for the Kitparamutation
is lethal, while heterozygotes have distinctive white feet and tail tips. Breeding
these heterozygotes with wild-type mice results in some wild-type offspring
with white feet and tails. Proof of the role of RNA in this non-Mendelian form
of inheritance was shown through injecting RNA from the Kit heterozygotes
into fertilized mouse oocytes also recapitulated the phenotype .
Another example of
RNA-mediated epigenetic heritability is the recent finding that an RNA-mediated
antiviral response can pass down in C. elegans worms. This is based on the inheritance of the siRNA machinery,
showing that cytoplasmic inheritance of the RNAs was required for the trait to
be passed on . Over 80 generations inherited this trait, showing the long-lasting
effects of epigenetic inheritance in certain cases.
dsRNAs from food survive digestion and
regulate our genes
With all the studies mentioned
showing the wide-reaching effects of short dsRNAs in
mediating organism function and inheritance, it is critical within the context
of GM crops that utilize RNAi technology to assess
the possible exposure routes to humans as well as non-target organisms. Recent
work shows that miRNAs from plant foods survive digestion and even go on to
mediate genes in the body following consumption. A 2012 study analysed global
miRNA levels in humans and 5 other mammalian species following consumption of
rice. They found a selective uptake of 30 miRNAs and when they investigated
this further in mice models, they found that one of the miRNAs, mi168a went on
to mediate expression of the liver gene (LDLRAP1), leading the authors
to speculate whether dsRNAs are indeed a nutrient  (see  How Food Affects
Genes, SiS 53). Other studies have since confirmed the presence of
exogenous RNA from food in humans, including rice, corn, barley, tomato,
soybean, wheat, cabbage, grapes and carrot . Further, miRNAs have been
discovered in human plasma and other body fluids including human breast milk,
which is stable in conditions commonly assumed to degrade RNA including freeze
thaw cycle, high and low acid conditions (as would be found in the stomach),
boiling, and extended storage, all of which destroys synthetic RNA [21-24].
Monsanto tries to discredit work
detecting plant RNAs in humans
Monsanto have attempted to discredit Zhang’s
work, most recently in a petition to the USDA for deregulation of MON87411
which contains dsRNA directed against the DvSnfy7 gene of the Western
corn rootworm . DvSnfy7 is the corn rootworm’s version of the gene coding
for Snf7, a protein that is well-conserved from yeast to human and several
studies have revealed its role in endosomal sorting and additional functional
roles in biological processes such as viral budding, cell
division and regulation of gene transcription. Their petition cited a study led
by Witwer, which failed to detect plant miRNA . This study used two
animals only, compared to that of Zhang’s team, which included 10 women, 11 men
(plus pooled serum from 10 extra individuals) along with 6 animals of 5
additional mammalian species. The Witwer study only looked at 7 plant miRNAs,
whereas Zhang’s team looked at global RNA levels. Monsanto
cited a second study which again failed to detect plant RNA in mammals after
assessing only a few miRNAs . Further, a communication from Monsanto
published in Nature Biotechnology claims to have conducted their own
experiments and failed to detect exogenous rice miRNA
in mice . However, failure to detect anything is not proof that the
exogenous RNA is not present in the mouse. The easiest way to find nothing is
to do the experiment badly. As pointed out by Zhang in response to the
publication , their detection of plant miRNA even in rice, the positive
control, was well below the expected levels, and hence minimizes the chance for detecting anything in the
mice. The technical issues surrounding their quantitative PCR experiments,
including lack of appropriate controls and absence of raw data make it
impossible to judge the quality of the experiment and the findings. They
clearly know the implications of this work and are trying their best, as they have done in the past, to discredit any
scientist who exposes the potential dangers of their products.
I have submitted a comment to the USDA on the behalf of
ISIS pointing out the above limitations as well as the risks (see below) omitted
by Monsanto in their petition.
So what are the risks of consuming siRNA
All the work done on dsRNAs and the RNAi
pathway clearly indicate that any nucleic acids introduced into our foods, can
survive digestion and end up in our blood and organs. This means there is a
worrying possibility that these dsRNAs will interact with human genes and alter their expression, which may even be
passed down the generations. Previous work using RNAi technologies for
gene therapy experiments in mice found that with 49 different miRNAs introduced
into mice, 36 were severely toxic; 23 were lethal in every case, killing the
animals within two months, showing the potential for toxicity despite the
designing of sequence-specific dsRNAs. Bioinformatics tools show
lethality/toxicity of RNA-based technologies despite designing dsRNAs to specific
sequences, there is around a 10 % error rate, suggesting it is impossible to
create a dsRNA to target one specific gene (see  New GM Nightmares
with RNA, SiS 58) and  RNA
Interference “Complex and Flexible”, SiS 59) and ). MicroRNAs do not require
complete complementarity to their target mRNA sequence, they only need a match
of 7 bases in a row to bind, making the potential for off-target effects
unavoidable. Without assessing the potential for off-target effects, it makes
no sense whatsoever to approve such crops. It is impossible to predict the
effects that each individual dsRNA will have on any given organism.
There are also species-specific effects of RNAi, making it difficult to predict
their toxicity from one organism to another.
have the additional problem of expressing these transgenes in an artificial
manner, interfering with the natural processes of the fluid genome and in
different species. Further, as in the case of the GM wheat DIR093 generated to have altered starch
content, the RNA sequences that have been inserted are present with both the
matching and inverted repeat on the same strand, which does not occur naturally
(see  for summary of miRNA GM crop risks). So for regulators to
assert that as we eat RNAs in food all the time that RNAs are all safe to eat
is both ignorant and misleading. This could be said of proteins, but prions
disease has proved that we cannot make such assumptions, especially if it is
out of its natural context or not naturally occurring. Sequence-independent
effects are also common sources of toxicity for oligonucleotide therapeutics,
which is length-dependent with increasing toxicity at lengths about 30
nucleotides, well below the length of the dsRNA DvSnf7 (240 nucleotides
long) contained in MON87411 and DR093 [33, 34].
The effects of
any dsRNA in GM crops are completely unpredictable and could impact generations
to come. Without thorough testing of likely off-target
effects, novel DNA/RNA molecules, and sequence independent effects, we cannot
allow these crops to be approved. From the evidence existing to date, it
appears unlikely that RNAi can really be specific to one intended target of one
particular species. Keeping up to date with the ever evolving field of
epigenetics is of upmost importance in
identifying such risks.
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Nina Galang Comment left 29th August 2014 13:01:05 Dear Mae Wan-Ho,
Thank you for your very informative and educational explanations and your continuing work on GMOs.
i wrote sometime ago that i would like to purchase one of your 'black' paintings. i was answered that you were out of the country and you would get back to me when you returned. Do you still have paintings that have not been bought?
Nina (Miriam College, Philippines)
Ken Lys Comment left 30th August 2014 11:11:39 The USA handing Monsanto a free pass to alter (and then own) the genome of EVERY food plant and food animal in the world is simply insanity. We are still easily decades away from understanding how life fully works through the DNA of an unaltered single species genome, and much further yet at understanding the long term implications of mixing genes cross-species. This is brand new technology and there has been enough discovered already to put a halt to this until our safety is assured - but that likley won't work given the profit Monsanto has already tasted and whatever US political motivations support this infant technology. Sadly, it will take a catastrophy in our environment or our health to wake us up.
Alice Cho Comment left 31st August 2014 07:07:26 What's the future of agriculture and food from Western Australia when its wheat & barley plant-breeding program (InterGrain) is now jointly owned by the state and federal governments and Monsanto? What's the chances of getting outcomes in the public interest from this commercial shareholding?