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. 2004 Aug 9;32(14):4237-43.
doi: 10.1093/nar/gkh764. Print 2004.

RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa

Agustin Chicas  1 , Carlo Cogoni, Giuseppe Macino
Affiliations

RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa

Agustin Chicas et al. Nucleic Acids Res. .

Abstract

RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions.

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Figures

Figure 1
Figure 1
Transposon relics derived siRNAs. RNA blots were hybridized with probes for the indicated regions. HMC17 is a relic of Tad, HMC38 is a maggy relic and HMC48 is a gypsy relic. The low-molecular-weight RNAs were extracted from a wild-type strain or from the indicated mutants. qde-1 is a mutant in the RdRP, qde-2 is a mutant in the PPD protein, qde-3 is a mutant in the putative RecQ helicase and dcl-1/dcl-2 is a double mutant in the two Neurospora Dicer genes.
Figure 2
Figure 2
The Neurospora RNAi machinery is not required for loci-specific Lys9H3 methylation. DNA co-immunoprecipitated with anti-trimethyl antibodies was quantified by real-time PCR. The relative values were obtained by dividing the values obtained for the HMCs by the values obtained for the wc-1 gene, which were extrapolated from individual external standard curves made with serial dilutions of input DNA. The error bars represent the standard deviation of two different immunoprecipitation carried out with two different antibodies and analyzed in duplicates.
Figure 3
Figure 3
Transcripts derived from repeated sequences are targets of the Neurospora RNAi machinery. (A) RT–PCR analysis showing the presence of bidirectional transcripts derived from repeated sequences. (B) Quantification of pund transcripts in wild type and mutants in genes of the Neurospora RNAi pathway. The error bars represent the standard deviation of an RT reaction analyzed in triplicates.
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References

    1. Denli A.M. and Hannon,G.J. (2003) RNAi: an ever-growing puzzle. Trends Biochem. Sci., 28, 196–201. - PubMed
    1. Chicas A. and Macino,G. (2001) Characteristics of post-transcriptional gene silencing. EMBO Rep., 2, 992–996. - PMC - 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. Hammond S.M., Boettcher,S., Caudy,A.A., Kobayashi,R. and Hannon,G.J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science, 293, 1146–1150. - PubMed
    1. Volpe T.A., Kidner,C., Hall,I.M., Teng,G., Grewal,S.I. and Martienssen,R.A. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science, 297, 1833–1837. - PubMed

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