Raf1 Is the DCAF of the CLRC Complex Figure 5. Raf1 mutations disrupt interaction with Rik1. (A) Yeast two-hybrid assay. Interaction of Raf1 with Rik1 is indicated by growth on -Leu, -Trp, -His, -Ade plates. BD and AD: GAL4 Binding or Activation Domain fusions, respectively. (B) Western of FLAG-Raf1, FLAG-Raf1-R518A FLAG-Raf1- R576A and FLAG-Raf1-1 IPs analysed for FLAG-Raf1 and Rik1-Myc (right: cells grown at 36uC). (C) Western of FLAG-Raf1, FLAG-Raf1-R518A FLAG-Raf1- R576A and FLAG-Raf1-1 IPs analysed for FLAG-Raf1 and Clr4-Myc (right: cells grown at 36uC). (D) FLAG-Raf1, FLAG-Raf1-R518A, FLAG-Raf1-R576A and FLAG-Raf1-1 ChIP. Diagram shows cen1 primers used (black bar). Enrichment of FLAG-tagged proteins or untagged control were analysed by qPCR of otr relative to act1+(bottom: cells grown at 36uC). Error bar SD. (E) Westerns of Raf2-TAP IP analysed for FLAG-Raf1, FLAG-Raf1-1, FLAG-Raf1-R518A and FLAG-Raf1-R576A (right: cells grown at 36uC). doi:10.1371/journal.pgen.1002499.g005 Figure 6. The CLRC complex couples H3K9me and siRNA production. (A) Top panel: The CLRC complex is an active E3 ligase. The DCAF Raf1 binds a specific substrate (X) allowing its ubiquitination. This is essential for methylation of H3K9. Bottom panel: The CLRC complex is not an active E3 ligase. Cul4, Rik1, Raf1 and Raf2 form a protein scaffold allowing the correct targeting of the histone methyltransferase Clr4. (B) Individual CLRC components stimulate processing of centromeric transcripts into siRNAs independently of CLRC complex integrity. Raf1-Raf2, methylation of Mlo3 or other substrates and Rik1 (left) are required for processing centromeric transcripts into siRNAs. doi:10.1371/journal.pgen.1002499.g006 PLoS Genetics | www.plosgenetics.org 11 February 2012 | Volume 8 | Issue 2 | e1002499