Rhiana Lau MMG C174 Professor Simpson

1. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cellsScott 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.

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.

8. 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.

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.

10. 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).

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.

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.