WHAT'S HOT IN... BIOLOGY , Mar./Apr. 2008

MiroRNAs (miRNAs) are small pieces of non-coding single-stranded RNA that play a vital role in many biological processes by regulating the expression of genes. More than 5,000 different miRNAs have been discovered; the latest catalogue issued by the Wellcome Trust Sanger Institute lists 5,395. MiRNAs are clearly involved in some diseases, and equally clearly influence the expression of genes, but determining their effects more precisely has not been easy.

For one thing, each miRNA is believed to interact with up to 200 genes. For another, the genes that code for miRNAs are much bigger than the miRNAs and are often clustered close together or else spread across several introns (non-coding regions) of other genes. That makes a genetic approach to knocking out miRNA function all but impossible.

The paper at #10 adopts an entirely different approach and shows that it works. Markus Stoffel, then at Rockefeller University in New York (and currently at ETH Zurich, Switzerland), and his colleagues synthesized RNA complementary to an miRNA but chemically modified and joined to a cholesterol molecule. In this they were mimicking the structure of synthetic silencing RNAs (siRNAs), which are double-stranded sequences designed to attach to and silence messenger RNA, thus effectively silencing the gene that codes for the mRNA. (Thomas Tuschl, a co-author of the miRNA paper, led the team that engineered synthetic siRNAs to give them "drug-like properties" such as stability and the ability to get inside cells.)

Stoffel's team homed in on miR-122, an abundant miRNA that is specific to liver cells. They made what they called an antagomir--antagomir-122--and injected it into mice. Levels of miR-122 plummeted. Unmodified anti-122 had no effect, while chemically modified anti-122 that was not linked to cholesterol had a partial effect. What had happened to the miR-122? The "proper" antagomir apparently prompted the destruction of miR-122, while the "partial" antagomirs bound to the miR-122, preventing it from doing its job but not actually removing it from the system.

MiRNAs have been implicated in many diseases, among them cancers, hepatitis, and diabetes, so there is great interest in these molecules as novel therapeutics. Stoffel's group examined several pharmacological properties of antagomirs, discovering that miR-122 remained undetectable up to 23 days after the antagomir was administered.

To examine which tissues would be affected they created an antagomir to miR-16, which is present in all tissues. Antagomir-16 suppressed miR-16 in all tissues except the brain. So although they are not able to cross the blood-brain barrier, antagomirs could silence their target miRs in all other tissues. Furthermore, the antagomirs were specific to their targets alone. MiR-192 and miR-194 are processed from a single precursor molecule; antagomir-192 blocks miR-192 and has no effect on miR-194, while antagomir 194 blocks miR-194 but not miR-192.

Individual miRNAs influence the expression of many target genes. Silencing an miRNA might therefore be expected to influence many genes too. Stoffel's team looked at gene expression in liver cells treated with antagomir-122. In all, 363 genes were significantly upregulated and 305 were downregulated.

Among the upregulated genes were many that are normally suppressed in liver cells, suggesting that miR-122 has a role in ensuring that adult liver cells remain differentiated as liver cells. Not all of the genes influenced by antagomir-122 will be under the direct control of miR-122, but an investigation of gene sequences revealed that a far higher proportion than expected contained the miR-122 target sequence. This means that there are probably more direct targets for each miRNA than had previously been thought.

In a similar vein, the miR-122 target sequence is distinctly lacking from those genes that are downregulated by the antagomir. The genes that were downregulated by antagomir-122 included at least 11 genes involved in the synthesis of cholesterol. Other antagomirs had no effect on the cholesterol pathway, showing that it is the antagomir sequence that matters, not the fact that it is bound to cholesterol.

All this is effectively proof of concept: it is possible to design and make an antagomir to a specific miRNA, and the antagomir will block that miRNA in vivo. That creates a powerful tool for investigating gene regulation and functioning, and even more powerful potential therapies for disease management.

Improved diagnosis too is on the cards, using miRNA profiles to indicate specific disturbances. A quick scan of the papers citing Krutzfeldt et al. at #10 reveals a roughly equal split between those interested in disease and those interested in basic biology. It is clear that miRNAs, discovered only in 1993 and named as recently as 2001, still have much to reveal about the workings of the cell in sickness and in health.

Dr. Jeremy Cherfas is Science Writer at Bioversity International, Rome, Italy.