Download An RNA-directed nuclease mediates post

An RNA-directed nuclease mediates post-transcriptional
gene silencing in Drosophila cells
Scott M. Hammond, Emily Bernstein, David Beach, and Gregory J.
Hannon
Rhiana Lau
MMG C174
Professor Simpson
Gene Silencing
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Gene silencing has been observed in many organisms: C.
elegans, Drosophila, planaria, hydra, trypanosomes, fungi, and
plants.
There are different mechanisms of gene silencing. Examples
include transgene cosuppression and posttranscriptional gene
silencing.
Studies correlated certain gene silencing processes to
methylation of promoter sequences and alterations in chromatin
structure.
RNA interference (RNAi) is considered a posttranscriptional
gene silencing process.
A common trigger for these processes is RNA.
Double-stranded RNAs are most effective at triggering silencing
of gene expression.
Gene Silencing
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Biological functions for RNAi include antiviral defense, genoprotective mechanisms, and regulation of cellular gene
expression.
Characteristics of RNAi
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Most studies with RNAi have been done in vitro using cell-free
extracts.
Upon treatment with dsRNA, a nuclease known as RISC (RNAinduced silencing complex) is assembled. RISC, a multiprotein
complex, is about 500 kDa.
This complex degrades target mRNAs homologous to the
dsRNA in a sequence-specific manner.
Small RNAs about 22 nucleotides long that were homologous to
the silenced gene were consistently identified in the extract.
These small RNAs cofractionated with the RNAi-effector
nuclease (RISC).
These results imply that these small RNAs function to guide the
enzyme complex to the substrate.
Characteristics of RNAi
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Substrate RNAs were degraded with a periodicity that matched
the size of the small RNAs.
An activity in extracts was also observed to process dsRNA
triggers into fragments about 22 nucleotides long.
These small RNAS were termed siRNAs (small interfering
RNAs).
Double-stranded RNA triggers processed into siRNAs by
enzyme in RNAse III family, specifically the Dicer family.
Dicer family proteins are ATP-dependent nucleases.
These proteins contain an amino-terminal helicase domain, dual
RNAse III domains in the carboxy-terminal segment, and
dsRNA-binding motifs. They can also contain a PAZ domain.
Characteristics of RNAi
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The PAZ domain is a motif also found in Argonaute proteins,
which have recently been found to bind Dicer.
 It is hypothesized that Argonaute proteins within RISC recruit
Dicer, thus enabling the incorporation of siRNAs into RISC.
Figure 1.
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Transiently transfected Drosophila S2 cells with lacZ expression
vector in order to visualize -gal activity (blue cells).
Co-transfection with lacZ dsRNA (first 300 nucleotides of sequence)
led to reduced activity.
Co-transfection with control CD8 dsRNA or no dsRNA had little effect
on activity.
Therefore, dsRNA interferes with gene expression in cultured cells in a
sequence specific manner.
Figure 1.
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FACS (fluorescence-activated cell sorter) analysis was used to
determine whether RNA interference could target endogenous gene
expression.
S2 cells were transfected with double-stranded Drosophila cyclin E
RNA.
S2 cells were also transfected with lacZ dsRNA as a control.
Transfection with cyclin E dsRNA caused G1-phase cell cycle arrest,
demonstrating that RNAi did indeed target endogenous genes.
They determined that the interference was length-dependent. Longer
dsRNAs were more effective than shorter dsRNAs.
Figure 1.
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They demonstrated that a common characteristic of RNAi is the
reduction of endogenous mRNA levels that are homologous to the
dsRNA transfected into the cells.
The gene fizzy is a component of the anaphase-promoting complex,
which is essential for ubiquitin-mediated proteolysis of anaphase
inhibitors. Cyclin A is essential during S, G2, and M phase of the cell
cycle.
Reduced expression of the corresponding mRNAs to the dsRNA
transfected into the cells was visualized by Northern blot.
Figure 2.
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S2 cells were transfected with either cyclin E or lacZ dsRNAs. The
cellular extracts were then incubated with synthetic cyclin E or lacZ
mRNAs and the results visualized by Northern blot.
The appropriate homologous transcripts were degraded in the extracts
containing the corresponding dsRNA.
The amount of degradation increased with time.
Therefore, the degradation of the target mRNAs occurs through
generation of a sequence-specific nuclease activity, otherwise known
as RISC (RNA-induced silencing complex).
Figure 2.
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The substrate requirements for nuclease activity were studied using
various cyclin E-derived transcripts. These transcripts were incubated
with S2 cells that had been transfected with cyclin E dsRNA.
RNAi nuclease activity more effectively degrades mRNAs with a
longer region of homology to the dsRNA.
The degradation of the mRNAs were specific to those containing
homologous sequences to the cyclin E dsRNA.
Figure 2.
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The sequence-specific nuclease activity was tested on antisense mRNA
substrates of differing lengths.
The antisense cyclin E mRNAs were also degraded in a lengthdependent manner. The transcripts with more homology to the
transfected dsRNA were degraded more efficiently.
From the experiments, mRNAs need to contain at least about 200
nucleotides of homologous sequence to the targeted region.