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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

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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 [1] New GM Nightmares with RNA, SiS 58). 

Investigations 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.

1. 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

2. Nucleotides paired outside the seed region stabilize interactions but do not influence miRNA efficacy

3. Functional miRNA targets are located close to the extremes of the 3’ (tail end) UTRs (untranslated regions) of protein-coding genes.

But many 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 [2]: “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.”

Human cells 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 rule-based interactions.

While 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 miRNAs.

The 18 514 mi-RNA-mRNA interactions representing 399 different miRNAs and 6 959 different protein-coding genes have been analyzed in detail.

Many 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 [2]: “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?


  1. Ho MW. New GM nightmares with RNA. Science in Society 58.
  2. 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 Edinburgh

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