There are numerous
applications for techniques that can reduce gene-specific expression
in mammalian cells. These range from reverse genetic experiments
(where the function of a gene is deduced from a reduction in its
activity) to potential uses in human therapy (for example, as a means
of suppressing the activity of deregulated oncogenes).
To date, mammalian gene suppression has
been achieved primarily via antisense RNAs. These RNA molecules
probably act by binding to complementary mRNA sequences, preventing
ribosome assembly or movement along a specific message, and thus
stopping translation. However, antisense RNAs do not always work
well in practice. This may be because some antisense RNAs cannot
bind to folded regions of the target mRNA.
Now a second method, RNA interference (RNAi),
is emerging as an effective tool for suppressing gene expression
in mammalian cells. Ambion has been following the research in this
area for some time. In the August 14, 2001 issue of The Proceedings
of the National Academy of Sciences, USA, Caplen and colleagues
showed that injection of short dsRNAs into mammalian cells or C.
elegans can cause degradation of the corresponding mRNA. The
authors successfully used this RNAi technique to silence 2 reporter
genes in 3 different mammalian cell lines. This technique may potentially
be expanded to silence a wide array of mammalian genes in a broad
range of cell lines.
The Effect of Long dsRNAs on Mammalian Gene Inhibition
RNAi is a post-transcriptional mode of
gene silencing. This phenomenon is characterized by the observation
that injection of dsRNA into a variety of organisms leads to
degradation of the corresponding mRNA. In C. elegans and Drosophila cells
it has been shown that these dsRNAs are broken down into ~21-25
nt fragments, termed short interfering RNAs (siRNAs), that then
bind to and tag the complementary portion of the target mRNA
for nuclease digestion.
While the natural presence of RNAi has
been observed in a variety of organisms (plants, protozoa, insects,
and nematodes), evidence for its existence in vertebrate cells
had been lacking. Transfection of long dsRNAs into mammalian
cells results in nonspecific gene suppression, as opposed to
the gene-specific suppression seen in other organisms. This suppression
has been partially attributed to a cascade that involves the
activation of protein kinase PKR, phosphorylation and inactivation
of the initiation factor eIF2a, and nonspecific translational
inhibition.
The Effect of Short Interfering RNAs (siRNAs) on
Mammalian Gene Inhibition
On the other hand, it had also been known
that dsRNAs less than 30 nt in length do not activate the PKR
kinase pathway described above. This observation prompted Caplen
and colleagues to test whether siRNAs less than 30 nt long can
cause gene-specific silencing in mammalian cells and C. elegans.
The authors tested their hypothesis by
monitoring the expression of 3 genes after siRNA transfection, unc-22 (in C.
elegans), gpf, and cat (in mammalian cells).
21-27 nt siRNAs corresponding to each of the 3 genes were synthesized.
Each siRNAs contained a 5' phosphate, a 3' hydroxyl group, and
a 2 nt 3'overhang. These siRNAs were introduced into C. elegans and
into the mammalian cells MEF, 293, and HeLa. Upon transfection,
expression levels of the corresponding genes were monitored using
several methods. Suppression of unc-22 gene, which encodes
a striated muscle component, was determined by the percentage
of nematodes with a twitching phenotype. Fluorescence intensity
and the number of GFP-positive cells were used to determine gfp suppression.
An ELISA-based assay was used to monitor a reduction in cat expression
levels.
Using these assays, the authors were able
to show that all 3 siRNAs were able to suppress the expression
of the corresponding genes. Moreover as expected, suppression
was taking place at the RNA level. Using Northern blots and mRNA
from gfp-transfected MEK cells, the authors demonstrated
that gpf siRNA caused a reduction in gfp, but not
in neo, mRNA levels.
Is RNAi a Universal Phenomenon in Mammalian Cells?
While only a limited number of genes and
cells have been tested, there is reason to believe that gene
silencing through siRNAs is a universal phenomenon in mammalian
cells. Namely, a number of genes that are involved in plant and
invertebrate siRNA formation have been conserved in mammalian
genomes. These include RNase III, an RNA-dependent RNA polymerase,
and the RDE/Argonaute family members. The authors speculate that
the mammalian RNAi genes may be involved in host defense (e.g.
viral RNA degradation) or gene regulation (e.g. imprinting).
siRNAs may prove to be more effective at
gene-specific suppression than antisense RNAs. The authors, for
example, were able to achieve a 2-3 fold higher level of cat and gpf inhibition
when using siRNAs. This higher potency may be due to the greater
stability of siRNAs. Alternatively, it may be due to the high
efficiency of target RNA degradation by the RNAi machinery.
Reference
Caplen, N.J., Parrish, S., Imani, F., Fire,
A., Morgan, R.A. (2001) Specific inhibition of gene expression
by small double-stranded RNAs in invertebrates and vertebrate systems. Proc.
Natl. Acad. Sci. USA 98: 9746-9747.
For more information about RNAi, see our review "RNA
Interference and Gene Silencing".
Related Articles
|