ISIS Report 22/05/13
RNA Interference "Complex and Flexible"
A technique hailed as a triumph in ‘precision
genetic engineering’ now proven to be nothing like that at all Dr Mae-Wan Ho
Announcing a new Report from ISIS. The most complete up-to-date summary of the dangers of GM agriculture in 52 pages. Buy Now, or download here
It has been known at least since 2003 that
RNA interference (RNAi) - a process whereby a short regulatory RNA sequence of
7 to 21 nucleotides can block the expression of (silence) specific genes – is
not as specific as it was touted to be, which is why applying it for genetic
modification is fraught with danger (see  New GM Nightmares
with RNA, SiS 58).
over the past decade have produced a set of ‘canonical rules’ governing the
interaction between the short regulatory microRNAs (miRNAs) and their target
mRNAs as follows.
Interactions are mediated by the ‘seed’ region,
a 6-8 nt long fragment at the 5’ end (head) of the miRNA that forms perfect (Watson-Crick)
base pairs with the target
Nucleotides paired outside the seed region
stabilize interactions but do not influence miRNA efficacy
Functional miRNA targets are located close to
the extremes of the 3’ (tail end) UTRs (untranslated regions) of
exceptions to the rules have also been uncovered recently - bulged nucleotide
interactions (due to non-paired bases); wobble G-U pairing (instead of A-U or
G-C); lack of seed pairing, with multiple mismatches, bulges and wobbles; G
bulge in the target; miRNA-binding at the 5’ UTRs of mRNA and within the coding
sequences; and binding within non-coding RNAs arising from pseudogenes - all of
which is summed up by a team of researchers at Edinburgh University in the UK
: “Together these data indicate that miRNAs can bind to a wide variety of
targets with both canonical and noncanonical base pairing, and indicate that
miRNA targeting rules may be complex and flexible.”
express more than 1 000 miRNAs, each potentially binding to hundreds of mRNAs,
only a small fraction of which has been identified experimentally. The research
team has now used a technique specially developed for capturing the miRNA bound
to their targets, cross-linking them and then sequencing the base-paired
miRNA-target RNA duplexes. They found that the exceptions far outnumber the
binding of most miRNAs includes the 5’ seed regions, around 60 % of seed
interactions are non-canonical (not according to the rules). They contain
bulged or mismatched nucleotides. Moreover, seed interactions are generally
accompanied by specific, non-seed base pairing. Only around 37 % of seed
interactions involve uninterrupted Watson-Crick base-pairing. Some 18 % of
miRNA-mRNA interactions involve the non-seed tail end of the miRNA, with little
evidence for contacts at the head seed end. MiRNA species systematically differ
in their target RNA interactions, and strongly overrepresented motifs are found
in the interactions sites of several miRNAs.
Most miRNA targets are mRNAs, comprising 70 % of the interactions;
other targets include pseudogenes and inter-intergenic non0coding (nc)RNAs, plus
substantial numbers of ribosomal RNAs, transfer RNAs, small nuclear RNAs and
The 18 514 mi-RNA-mRNA interactions representing 399 different
miRNAs and 6 959 different protein-coding genes have been analyzed in detail.
characterized miRNA interactions involve perfect complementarity between miRNA
5’region, particularly nucleotides 2-8 (seed sequence) and the target RNA.
Compared to random sequences, the data show strong enrichment for exact
(Watson-Crick, “canonical seed”) and near-exact (G-U pairs, up to one mismatch
or bulge “non-canonical seed”) seed matches. But non-canonical seed
interactions are ~1.7 times as common as perfect base pairing.
The identified miRNA target sites are markedly conservation
relative to flanking regions in an analysis of 46 vertebrate genomes,
supporting their biological importance. Regions of highest conservation are
typically the seed element (nt 1-8) and a downstream region (nt 13-19).
A mathematical (K means) clustering
analysis separated five classes of interactions according to distinctly
different base-pairing patterns. Class I-III interactions all involve the seed
regions; whereas class 1 (19 %) interactions are confined to the seed region, class
II and III additionally involve miRNA nucleotides 13-16 and 17-21 respectively.
Class IV (16%) binding is limited to a region in the middle and the tail end of
the miRNA and class V involves distributed or less stable base-pairing. Evolutionary
conservation and target down-regulation are strongest in class II. Two-thirds
of all miRNAs analyzed show nonrandom distribution across the five base pairing
classes. Most miRNAs are enriched in the seed interacting classes I-III, but
others showed highest enrichment in the non-seed class IV. There is a
prevalence of non-seed interactions overall.
The results are
verified by transfection experiments, for example, by inserting potential
target sequences into 3’ UTR of luciferase to observe down-regulation of
luciferase for the miR-92a seed and/or 3’ binding regions.
But that’s not the end of
the story. The results have been obtained in human embryonic kidney (HEK) cells
grown in culture; they do not apply to other cells or tissues in different
environments. The researchers comment on closing : “More generally, the spectrum of miRNA-mRNA
interactions is expected to rapidly change during differentiation, and viral
infection and following metabolic shifts or environmental insults.”
How can anyone still think
it is safe to apply RNAi in genetic modification?
- Ho MW. New GM nightmares with RNA. Science
in Society 58.
- Helwak A, Kudla G, Dudnakova T and
Tollervey D. Mapping the human miRNA Interactome by CLASH reveals frequent
noncanonical binding. Cell 2013, 153, 654-65. MRC Human Genetics
Unit, Institute of Genetics and Molecular Medicine, University of