1. Rhiana Lau MMG C174 Professor Simpson
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

2. Gene Silencing
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.

3. Gene Silencing
Biological functions for RNAi include antiviral defense, geno-protective mechanisms, and regulation of cellular gene expression.

4. Characteristics of RNAi
Most studies with RNAi have been done in vitro using cell-free extracts.
Upon treatment with dsRNA, a nuclease known as RISC (RNA-induced 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.
This description of the mechanism of RNAi as it is understood more recently than the paper we are discussing was published.

5. Characteristics of RNAi
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.

6. Characteristics of RNAi
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.

7. Figure 1.
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.
Visualization of -gal activity was through an in situ assay where the cells are fixed and then substrate is added to allow blue color to form.
ssRNA was also co-transfected in of either sense or antisense orientation. They also showed no effect on -gal activity.

8. S2 cells were also transfected with lacZ dsRNA as a control.
Figure 1.
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.
FACS analysis is based on flow cytometry, which is a technique that identifies different cells by measuring the light they scatter, or the fluorescence they emit as they flow through a laser beam.
Cyclin E is a gene that is essential for cell cycle regulation. It necessary for progression through the G1 phase to S phase of the cell cycle. The G1 phase is the initial growth phase, while the S phase is when DNA duplication occurs. Cyclin E associates with a cyclin-dependent kinase 2 and activates this kinase to phosphorylate genes essential during S phase.
Gating refers to configuring the computer to display the fluorescence signals from particles with a specific set of scatter properties. Normally during this process, the objective is to study single, living cells. Single, living cells scatter differently than dead or clumped cells, even after fixing. This paper in particular, however, does not mention what parameters they were gating for. It is probably safe to assume that they were selecting for single, living cells.
The Y-axis measures the amount of cells counted that were effectively transfected, which was visualized through GFP. The X-axis measures the amount of DNA in the cells, which was determined using propidium iodide dye.
The first peak in the no dsRNA transfected sample roughly corresponds to G1 phase, the trough to S phase, and the second peak to G2 phase.
As you can see, there isn’t much difference between the array of the no dsRNA transfected and the lacZ samples. The cells transfected with cyclin E dsRNA show effective G1 phase arrest seen by the one prominent peak, corresponding to G1 phase.

9. Figure 1.
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.
Explain figure.
The smear towards the bottom of the gels blotted with the cyclin E and cyclin A probe represent the degraded mRNA.
The lanes transfected with a dsRNA not corresponding to the probe show no degradation of the mRNA.

10. The amount of degradation increased with time.
Figure 2.
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).
The cyclin E mRNA was degraded over time in the extracts containing the cyclin E dsRNA, while the lacZ mRNA in the cyclin E extracts remained stable. The lacZ mRNA was degraded over time in the lacZ extract, while the cyclin E mRNA remained stable in the lacZ extracts.

11. Figure 2.
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.
The diagram at the bottom of the figure depicts the positions that the different mRNA substrates were synthesized from the cyclin E gene. The cross-hatched area indicates the extent of the cyclin E dsRNA sequence. Arrows pointing to the right indicate sense strands.
The lacZ samples were used as negative controls to demonstrate the sequence specificity of the nuclease activity.
The level of degradation decreases as the mRNAs get shorter.
E-ds samples refer to cyclin E dsRNA identical to the dsRNA transfected into the cells being added to the extract as substrate. Some degradation of the dsRNA results in the smear depicted in the gel.

12. Figure 2.
The sequence-specific nuclease activity was tested on antisense mRNA substrates of differing lengths.
The antisense cyclin E mRNAs were also degraded in a length-dependent 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.
Again, lacZ mRNA samples were added to cellular extracts as a control to demonstrate the sequence-specificity of the nuclease activity.