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Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

Nature Structural & Molecular Biology volume 22, pages 328–335 (2015)Cite this article

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Abstract

Argonautes and their small-RNA cofactors form the core effectors of ancient and diverse gene-silencing mechanisms whose roles include regulation of gene expression and defense against foreign genetic elements. Although Argonautes generally act within multisubunit complexes, what governs their assembly into these machineries is not well defined. Here, we show that loading of small RNAs onto Argonaute is a checkpoint for Argonaute's association with conserved GW-protein components of silencing complexes. We demonstrate that the Argonaute small interfering RNA chaperone (ARC) complex mediates loading of small RNAs onto Ago1 in Schizosaccharomyces pombe and that deletion of its subunits, or mutations in Ago1 that prevent small-RNA loading, abolish the assembly of the GW protein–containing RNA-induced transcriptional silencing (RITS) complex. Our studies uncover a mechanism that ensures that Argonaute loading precedes RITS assembly and thereby averts the formation of inert and potentially deleterious complexes.

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Figure 1: ARC subunits Arb1 and Arb2 are required for RITS assembly.
Figure 2: Loading of small RNAs onto Ago1 is required for assembly of RITS but not ARC.
Figure 3: ARC binds small RNAs in vivo that bear features of Dcr1-generated duplexes.
Figure 4: Immunopurified Ago1 binds duplex small RNAs in vitro in an Arb1-dependent manner.
Figure 5: Arb1 is required for all Ago1 small RNA–loading activity in vivo.
Figure 6: ago1+ overexpression partially suppresses silencing defect of arb1Δ and arb2Δ by overcoming the requirement for ARC in small-RNA loading.
Figure 7: Regulation of RITS-complex assembly by small-RNA loading onto Argonaute.

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Acknowledgements

We thank members of the Moazed laboratory for helpful discussions; E. Gerace, M. Halic, N. Iglesias and J. Xiol for advice on protocols; M. Halic and R. Yu for scripts and advice on bioinformatic analysis; C. Centrella for technical assistance; and E. Egan, N. Iglesias, R. Jain, J. Xiol and R. Yu for critical reading of the manuscript. This work was supported by the US National Science Foundation Graduate Research Fellowship Program (D.H.) and the US National Institutes of Health grant R01 GM072805 (D.M.). D.M. is supported as a Howard Hughes Medical Institute Investigator. D.H. dedicates this paper to the memory of his beloved father, George Holoch, who died on 9 April 2013.

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  1. Department of Cell Biology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Daniel Holoch, Danesh Moazed & Danesh Moazed

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  1. Daniel Holoch
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D.H. and D.M. designed the study. D.H. performed all experiments and bioinformatic analysis. D.H. and D.M. analyzed the data and wrote the paper.

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Correspondence to Danesh Moazed.

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Integrated supplementary information

Supplementary Figure 1 Ago1 associates with Arb1 and Arb2 independently of Tas3.

Mixture mass spectrometry analysis of the indicated FLAG purifications. Uniquely detected peptides, including overlapping peptides, are reported for each protein in each purification. Percent coverages shown are calculated by dividing the total number of amino acid residues represented in any peptide for a given protein in a given purification by the protein’s total length.

Supplementary Figure 2 Ago1 D580A, Ago1 F276A R773E, Ago1 F276A and Ago1 R773E are null-mutant proteins whose small RNA–binding activity correlates with their assembly into RITS.

(a) Schematic illustrating the ectopic insertion of 3xFLAG-tagged alleles of ago1 with endogenous promoter and terminator sequences near the trp1+ locus on chromosome 2. The native ago1+ locus harbors either a wild-type untagged allele or a deletion. (b) Tenfold serial dilutions of otr1R::ura4+ pericentromeric silencing reporter cells of the indicated genotypes, plated on non-selective medium or medium containing 5-FOA. (c) Northern blot analysis of dg siRNAs in total small RNA fractions and RNA immunoprecipitated with wild-type and mutant 3xFLAG-Ago1 proteins, in cells also expressing untagged wild-type Ago1, and Western blot analysis of input extracts and FLAG-immunoprecipitated material. (d) Western blot analysis of co-immunoprecipitation experiment to assay association of Tas3 with the indicated Ago1 proteins. (e) Western blot analysis of co-immunoprecipitation experiment to assay association of Arb1 with the indicated Ago1 proteins.

Supplementary Figure 3 RITS and ARC bind populations of pericentromeric small RNAs with similar sequences.

Tracks showing the normalized numbers of reads mapping to the dg and dh repeats flanking the centromere of chromosome 1, for small RNAs co-purifying with Tas3-TAP (RITS) or TAP-Arb1 (ARC), excluding tRNAs.

Supplementary Figure 4 Arb2 is not required for in vitro loading of duplex small RNAs onto immunopurified Ago1.

(a) Western blot analysis of input fractions and IgG magnetic beads after one-step purification of RITS and ARC from dcr1Δ cells expressing TAP-tagged subunits Tas3 or Arb1 or no tagged protein. (b) Phosphorimager scan of a non-denaturing polyacrylamide gel showing the 5’-end-labeled single-stranded and annealed duplex small RNAs used in in vitro binding assays. (c) Western blot analysis of the beads from FLAG purifications from the indicated cells, in aliquots equal to one-eighth of those used in the binding assay. (d) Phosphorimager scan of eluted RNAs after in vitro binding to immobilized 3xFLAG-Ago1 purified from the indicated wild-type and mutant cells. (e) Quantification by densitometry of the results shown in (d).

Supplementary Figure 5 Overexpressed Ago1 suppresses arb1Δ and arb2Δ not simply by rescuing protein stability and does not suppress other pericentromeric silencing mutants.

(a) Western blot analysis of total protein prepared from arb1Δ and arb2Δ cells expressing 3xFLAG-ago1 either from the endogenous locus or from an overexpression plasmid. Relative quantity of total protein loaded is indicated for each lane. Red pixels indicate saturated signal. (b) Western blot analysis of total protein prepared from cells of the indicated genotypes. Relative quantity of total protein loaded is indicated for each lane. (c) Tenfold serial dilutions of otr1R::ura4+ pericentromeric silencing reporter cells of the indicated genotypes, transformed with an empty vector (denoted with a “–”) or a 3xFLAG-ago1 overexpression plasmid (denoted with a red “+”), plated on non-selective medium or medium containing 5-FOA. Similarly, tenfold serial dilutions of otr1R::ade6+ cells of the indicated genotypes plated on medium containing a limiting concentration of adenine.

Supplementary Figure 6 Ago1 L317A protein does not complement ago1Δ but does not show a significant reduction in loading of pericentromeric siRNAs.

(a) Tenfold serial dilutions of otr1R::ura4+ or imr1R::ura4+ pericentromeric silencing reporter cells of the indicated genotypes plated on non-selective medium or medium containing 5-FOA. (b) Western blot analysis of whole cell extracts and FLAG immunoprecipitates prepared from wild-type cells transformed with the indicated 3xFLAG-ago1 overexpression plasmids, and Northern blot analysis of RNA extracted from each immunoprecipitated sample.

Supplementary Figure 7 ago1 D580A and ago1 F276A R773E are dominant-negative alleles when overexpressed.

Tenfold serial dilutions of otr1R::ura4+ pericentromeric silencing reporter cells of the indicated genotypes, transformed with an empty vector or a 3xFLAG-ago1 overexpression plasmid (wild-type or mutant as noted), plated on non-selective medium or medium containing 5-FOA.

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Holoch, D., Moazed, D. Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex. Nat Struct Mol Biol 22, 328–335 (2015). https://doi.org/10.1038/nsmb.2979

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