Cancer Research - Europa

SIROCCO
Silencing RNAs: organisers
and coordinators of complexity
in eukaryotic organisms
Summary
RNA silencing is the natural ability of a cell to turn off genes.
Only a few years ago it was unknown, but now RNA silencing
is one of the most powerful tools available to researchers.
Recent discoveries have revealed a previously unknown role
for RNA (ribonucleic acid). They have shown how, in addition
to the previously understood role as a cellular messenger
that directs protein synthesis, RNA can also silence expression
of genes. By introducing specifi c silencing RNAs into an
organism, the expression of genes can be turned down in
a controlled way. The phenomenon of RNA silencing is
thought to have evolved as a defence mechanism against
viruses. In primitive cells it was a type of immune system that
could recognize and then silence viral genes. Later in evolution
the silencing mechanism was recruited for switching off
genes involved in normal growth of cells and responses to
stress. Small regulatory RNAs (sRNAs) are the mediators of
RNA silencing and are important integrators of genetic, epigenetic
and other regulatory systems. They are the focus of
the SIROCCO programme. sRNAs have been referred to as
the dark matter of genetics: a recently discovered mass of
molecules that crucially aff ect the behaviour of the genetic
universe through interactions at the RNA level.
Problem
The exploitation of sRNAs off ers many opportunities for
improving the diagnosis and therapy of human disease and
for advances in biotechnology. sRNAs fall into two major
classes:
• short interfering RNAs (siRNAs) which are 21-24 nucleotide
RNAs derived from long double-stranded RNA;
• microRNAs (miRNAs) which are derived from transcripts
containing partially double-stranded stem-loop ‘hairpin’
structures about 70 nucleotides long. Both are cleaved
from their precursor RNA by double stranded RNA-specifi
c endonucleases. One strand of the resulting small RNA
is loaded into RNA-induced silencing complex (RISC) that
also contains Argonaute (AGO) proteins. Binding to the
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Keywords | RNA silencing | microRNA | RNA interference | short interfering RNA | developmental biology |
molecular biology | gene expression |
Aim
correct Argonaute protein is necessary for cleavage of
the target messenger RNA. siRNAs and miRNAs have
been found in a variety of organisms including plants,
fruit fl ies, zebrafi sh, mice, and humans. sRNAs are also
a useful tool in the laboratory, where they can be used to
silence gene expression (RNA interference).
The overall objectives of the SIROCCO project are:
• create catalogues of sRNAs from healthy and diseased
cells. Novel sRNAs will be identifi ed through using a combination
of bioinformatics and high throughput sequencing;
• determine the tissue- and cell-type pattern of miRNA
expression using microarray, RNA blot and in situ hybridisation
methods;
• fully refi ne methods for sRNA detection. These detection
methods will be enhanced using locked nucleic acidcontaining
and other oligonucleotide probes, and by
modifi ed PCR methods;
• characterise proteins and subcellular compartments re quired
for sRNA processing and activity. At present, there is a foundation
of knowledge about miRNAs, but very little is known
about siRNAs. Genetic, biochemical and imaging approaches
will be used to fully characterise the molecular machines
responsible for both miRNA and siRNA biogenesis;
• dissect sRNA regulatory networks. It is known that miRNAs
may aff ect particular target mRNAs but how their activity
fi ts into more complex regulatory networks is poorly understood.
Developing this understanding is one of the major
objectives of the SIROCCO programme;
• identify rules for sRNA effi ciency and specifi city. The RNAsilencing
effi ciency of sRNAs will be determined by assay
of sRNAs, their precursors or their DNA in transgenic organisms,
in cell cultures or in vitro;
• explore delivery methods for sRNAs or sRNA precursors.
Effi cient use of sRNAs as pharmaceuticals will depend on
the development of methods for their effi cient delivery into
cells and animals. Current technology uses modifi ed viruses
to introduce siRNAs into cells to reduce expression of a target
gene. In the later stages of the project, the SIROCCO
consortium will initiate research into the suppression of
genes implicated in various diseases.
The mechanism of RNA silencing must be thoroughly understood
in order to use RNA as a drug without side eff ects. It is
also necessary to understand more about the role of silencing
RNAs in normal growth and development. That information
will then allow us to use the presence of silencing RNAs to
diagnose disease states in a cell.
CANCER RESEARCH PROJECTS FUNDED UNDER THE SIXTH FRAMEWORK PROGRAMME