An RNA-directed nuclease mediates post-transcriptional An RNA-directed nuclease mediates post-transcriptional gene silencing in gene silencing in DrosophilaDrosophila cells cells

Scott M. Hammond, Emily Bernstein, David Beach, and Gregory J. Scott M. Hammond, Emily Bernstein, David Beach, and Gregory J. HannonHannon

Rhiana LauRhiana LauMMG C174MMG C174

Professor SimpsonProfessor Simpson

Gene SilencingGene 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.

Gene SilencingGene Silencing

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

Characteristics of RNAiCharacteristics 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.

Characteristics of RNAiCharacteristics 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.

Characteristics of RNAiCharacteristics 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.

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.

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.

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 1.

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.

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.

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.

Figure 2.