Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Oct 1;18(19):2359-67.
doi: 10.1101/gad.1218004. Epub 2004 Sep 15.

A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe

Alla Sigova  1 , Nicholas Rhind, Phillip D Zamore
Affiliations

A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe

Alla Sigova et al. Genes Dev. .

Abstract

The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Experimental strategy. The coding sequence of GFP was fused in frame with the adh1 locus on chromosome 3. The kanamycin resistance gene (aph) was inserted adjacent to the adh1:gfp transgene as an insertion marker under the control of the Ashbya gossypii translation elongation factor 1α gene promoter. Red arrows indicate the position of adh1:gfp-specific primers used in the quantitative RT-PCR assays in Figure 3. The silencing trigger was expressed from an episomal plasmid encoding a hairpin transcript corresponding to GFP expressed from the nmt1 promoter. The GFP dsRNA hairpin contained within the loop sequences the 67-bp intron 1 from rad9 to facilitate hairpin expression.
Figure 2.
Figure 2.
The GFP hairpin triggers adh1:gfp silencing. (A) Representative FACS data demonstrating that GFP silencing occurs only in the presence of the GFP hairpin. Silencing of GFP expression, measured by fluorescence intensity, was not observed for the empty vector alone, and silencing was lost when the silenced strain was grown in the presence of uracil to cause loss of the hairpin-encoding plasmid. Ten-thousand cells were analyzed for each genotype. See also Figure 5. (B) Representative FACS data demonstrating that GFP silencing is triggered by the GFP hairpin but not by either sense or antisense GFP transcripts.
Figure 5.
Figure 5.
GFP silencing depends on the RNAi machinery but not the transcriptional silencing proteins Chp1, Tas3, or Swi6. The geometric mean of fluorescence intensity was determined for each strain from experiments like that in Figure 2, and normalized to the geometric mean for the wild-type adh1:gfp strain. The data are the average ± standard deviation for three trials.
Figure 3.
Figure 3.
GFP silencing reflects a reduction in the steady-state level of adh1:gfp mRNA. The steady-state level of mRNA was measured for gfp and aph, relative to the expression level of the act1 mRNA. For each strain three independent cultures were assayed; each assay was analyzed by quantitative RT-PCR in triplicate. The data are presented as the average of the three independent trials (using the average value for the triplicate quantitative RT-PCR reactions) ± the standard deviation of the three independent trials.
Figure 4.
Figure 4.
GFP silencing triggered by the GFP hairpin is posttranscriptional. (A) Representative nuclear run-on experiment. (B) The average value ± standard deviation for three independent run-on experiments is presented. For each strain, the rates of transcription for both aph and adh1 were measured and normalized to the rate of transcription of act1. Firefly luciferase (Pp luc) served as a negative control. No decrease in transcription of adh1:gfp was observed when GFP was silenced by introduction of the GFP hairpin-expressing plasmid. The rate of aph transcription was essentially constant in all aph-containing genotypes. (C) RNA Pol II chromatin immunoprecipitation (ChIP). The density of RNA Pol II at the adh1:gfp locus was not decreased by the introduction of the GFP hairpin, reinforcing the view that silencing triggered by this hairpin is posttranscriptional. Data are the average of two independent trials. The data for adh1:gfp and those for aph were separately normalized to their respective values in the absence of the empty or hairpin vector.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aravin A.A., Naumova, N.M., Tulin, A.V., Vagin, V.V., Rozovsky, Y.M., and Gvozdev, V.A. 2001. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr. Biol. 11: 1017-1027. - PubMed
    1. Bahler J., Wu, J.Q., Longtine, M.S., Shah, N.G., McKenzie III, A., Steever, A.B., Wach, A., Philippsen, P., and Pringle, J.R. 1998. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14: 943-951. - PubMed
    1. Bernstein E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366. - PubMed
    1. Catalanotto C., Azzalin, G., Macino, G., and Cogoni, C. 2002. Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes & Dev. 16: 790-795. - PMC - PubMed
    1. Catalanotto C., Pallotta, M., ReFalo, P., Sachs, M.S., Vayssie, L., Macino, G., and Cogoni, C. 2004. Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol. Cell Biol. 24: 2536-2545. - PMC - PubMed

MeSH terms