Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast

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Abstract

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.

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Figure 1: Purification of Flag-Ago1 and identification of Arb1 and Arb2.
Figure 2: ARC contains siRNAs and is required for siRNA generation but does not localize to centromeric heterochromatin.
Figure 3: Immunofluorescence localization of ARC subunits.
Figure 4: Distribution of single-stranded and duplex centromeric siRNAs in the ARC and RITS complexes and inhibition of Ago1 slicer activity by Arb1.
Figure 5: The slicer activity of Ago1 is required for siRNA maturation and heterochromatin assembly.
Figure 6: The slicer activity of Ago1 is required for its localization to centromeric repeats and for the spreading of H3-K9 methylation.
Figure 7: Model for the roles of ARC and RITS complexes.

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Acknowledgements

We thank M. Siomi (University of Tokushima) for pGEX-5X-dAgo1, D. Patel (Sloan-Kettering Cancer Center) for plasmids and J. Buchberger, S. Colmenares and C. Morris for comments on the manuscript. This work was supported by grants from the US National Institutes of Health (D.M. and S.P.G.) and grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (T.I. and J.-I.N.). M.B. is supported by a European Molecular Biology Organization long-term fellowship. J.V. is supported by a postdoctoral fellowship from the Spanish Ministry of Education and Science. D.M. is a Scholar of the Leukemia and Lymphoma Society.

Author information

S.M.B., T.I., M.B. and J.V. carried out experiments; J.-I.N., S.P.G. and D.M. supervised research; S.B. and D.M. wrote the paper with input from all other authors.

Correspondence to Danesh Moazed.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Flag-Ago1 and Arb1-TAP are functional (PDF 2690 kb)

Supplementary Fig. 2

Physical association of Ago1 with Tas3 or Arb1 (PDF 40 kb)

Supplementary Fig. 3

Centromeric transcripts accumulate in arb1δ strain (PDF 114 kb)

Supplementary Fig. 4

Purification of proteins used for in vitro activity assays (PDF 151 kb)

Supplementary Fig. 5

Diagram of Arb1 homologs (PDF 891 kb)

Supplementary Table 1

Mixture mass spectrometry of Arb1-TAP–associated proteins (PDF 79 kb)

Supplementary Table 2

Spectral count normalized to Mw for mixture of proteins associated with Flag-Ago1 preparations (PDF 66 kb)

Supplementary Table 3

Strains used in this study (PDF 80 kb)

Supplementary Methods (PDF 108 kb)

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Buker, S., Iida, T., Bühler, M. et al. Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast. Nat Struct Mol Biol 14, 200–207 (2007) doi:10.1038/nsmb1211

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