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Monday June 7/Morning
SMALL RNAS II
Khalid Siddiqui
The recent discovery of
RNAi has made a huge contribution to our understanding of the mechanisms
of epigenetic regulation of gene expression, as well as providing a very
useful yet simple tool for basic research. The second session devoted to
small RNAs included various genetic screens in a hunt for other players
in the RNAi pathway as well as some mechanistic insights into how small
RNAs may play a role in gene silencing in metazoans.
The initial screen done
for RNAi mutants in Craig Mello’s group (U Mass Med Centre) had led to
the identification of the rde genes. It was discovered that these fell
into two classes – those that affected transposon silencing and those
that didn’t. Mello continued to characterize the latter and identified
the worm Argonaute homologs, some of which have been reported to have
roles in RNAi and interact directly with other known components of the
pathway. His group has been investigating the known piwi/paz domain
proteins and reported on one specific protein F20D12.1, the loss of
which is correlated with a defect in chromosome segregation and leads to
the speculation that the role of RNAi in centromere function may be
conserved in worms as well.
Ronald Plasterk (Hubrecht
Laboratory, Netherlands) discussed work on a set of mutants that lead to
increased mobility of transposons in C. elegans and showed that several
of these genes were allelic to the first class of rde mutants isolated
by Mello. The common feature of both these processes appears to be a
requirement for dsRNA mediated amplification at an early step in the
process that leads to efficient suppression. Silencing by RNAi was
correlated with histone modifications in these transposons resulting in
chromatin silencing, suggesting a conserved mechanism for dealing with
repeat elements from fission yeast to worms. Finally, data was presented
for a series of experiments, which demonstrated that perfectly paired
let-7 miRNA caused growth defects and that several unpaired positions in
the miRNA were necessary for normal development in zebrafish again
indicating distinct mechanisms for miRNA mediated silencing versus siRNA
mediated silencing effects.
A role for RNAi in
transposon silencing in plants was presented by Robert Martienssen (CSHL)
whose group has done extensive work in understanding the mechanism of
heterochromatin regulation in Arabidopsis. Analysis of mutants with
increased transposon mobility revealed two classes of genes that,
surprisingly, regulated different types of transposons by distinct but
interacting complexes or pathways. One class of genes included that
encoding a DNA methyltransferase and chromatin remodeling ATPase and
regulated the majority of transposons. A small but distinct subset of
repeat elements were silenced by histone modifying enzymes (H3K9 methyl
transferase) and genes in the RNAi pathway. It was suggested that
silencing marks on chromatin and DNA methylation produced by these
pathways were used to distinguish transposons from genes and that these
repeat elements could actually regulate the genes that they integrated
into. An interesting link was also established between RNAi mediated
silencing and genomic imprinting in plants by the demonstration that an
imprinted gene was expressed co-incident with the appearance of small
RNAs from a SINE element integrated into it.
A biochemical
investigation into the RNAi pathway was discussed by Greg Hannon (CSHL)
whose group has been characterizing a novel dsRNA-binding gene in flies
called “Gripper” that may be involved in the processing of miRNAs in the
nucleus. He presented data supporting the initial two-hybrid observation
that Gripper interacts with the Drosophila ribonuclease as part of a
larger “microprocessor” complex and co-purifies with small RNAs. In
addition, Hannon’s group has been doing a systematic knockout of the
recently identified Argonaute genes in the mouse, most of which result
in embryonic lethality. The miwi knockout in particular is viable but
leads to male sterility and raises the possibility that the RNAi pathway
may be involved in germline development. Analysis of Ago
immunoprecipitated from cellular extracts indicates that all the
homologs examined copurified with small RNAs, but only Ago2 containing
complexes could direct cleavage of the target messenger RNAs. This
indicates that the different Ago gene products are most likely
associated with distinct activities and may be involved in different
silencing pathways, perhaps distinguishing between the perfectly paired
siRNAs and the mismatch containing miRNAs, which are known to have
different effects on target mRNAs.
Gary Ruvkun (Harvard
Medical School) reported on several genetic screens in the nematode C.
elegans using GFP expression/repression as a marker. These screen were
aimed at identifying new genes involved in RNAi. An initial screen of
over 18,000 clones revealed about 33 genes involved in this pathway
including novel genes coding for RNA binding proteins, histone modifiers
and signal transduction proteins. In a separate screen, Ruvkun’s group
also identified proteins specifically involved in microRNA processing
and metabolism, a subset of which was non overlapping with the siRNA/RNAi
pathway. A third screen revealed a whole set of candidate regulatory
genes that enhanced RNAi phenotypes, including an unexpected gene
product with siRNase activity. The natural pathways of RNAi and miRNA
induced gene silencing thus appear to be far more complex than
previously imagined and involve networks of several gene products that
have distinct components for the regulated silencing of different
cellular processes.
The session ended with
an excellent demonstration of the potential of RNAi in mammalian genetic
screens by Rene Bernards (Netherlands Cancer Institute). Bernards
described three successful loss of function screens performed on
cultured human cells. A library consisting of pools of plasmids
expressing unique short hairpin RNAs was generated targeting nearly
8,000 human genes. With the help of sensitive assays for cell growth or
by employing luciferase reporter assays, these hairpins were used to
pinpoint genes that were potential targets of deubiquitinating enzymes
or that functioned as tumor suppressor genes or suppressors of TGF-
mediated growth arrest. These screens will be useful in identifying
potential therapeutic targets for small molecule drug design as well as
novel genes that would have been previously ignored for analysis using
conventional tools available. The availability of such hairpin RNA
libraries coupled with specific cellular assays will enable us to
perform genetics in mammalian cells with the same ease and confidence as
one would be able to using classical yeast genetics and greatly
accelerate our understanding of basic cellular processes.
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