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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.