With Cul4. doi:10.1371/journal.pone.0104161.graf2-S100F mutants at 36uC compared to wild-type cells (Figure 2D). As expected, the raf2-E104A allele did not affect Swi6 localisation. Disruption of centromeric heterochromatin is known to result in defective chromosome segregation as seen in clr4D and RNAi mutants [44,47]. In concordance with the observed defects in centromeric heterochromatin integrity, a high frequency of lagging chromosomes was evident in late anaphase raf2-I98A and raf2-S100F cells at 36uC, but not in raf2-E104A or wild-type cells (Figure 3). In summary, the above analyses indicate that the RFTS domain of Raf2 is important for the maintenance of heterochromatin integrity and thus centromere function.Odronextamab centromeres but is dispensable for the production of siRNA, as previously proposed [23,48].Mutations within the RFTS domain disrupt the interaction of Raf2 with CulThe function of the RFTS domain of mammalian DNMT1 has been the subject of several studies [27,32,39,49]. However, the role of RFTS domains in proteins such as Raf2 in organisms like S. pombe that lack DNA methylation remains unknown. As point mutations within the Raf2 RFTS domain affect heterochromatin integrity we tested whether these specific raf2 mutations affect the interactions between Raf2 and other components of CLRC. We therefore set up a targeted yeast-twohybrid (Y2H) assay to determine if the Raf2-I98A, Raf2-S100F and Raf2-E104 mutations affect interactions between Raf2 and other CLRC components. As previously described, we detect a direct interaction between full-length Raf2 and Cul4 (Figure 5A) [22] while interactions of Raf2 with Rik1, Raf1 and Clr4 could not be detected by Y2H.Anagliptin Our Y2H analyses demonstrate that the Raf2-I98A and Raf2-S100F mutations, but not the weak Raf2E104 mutation, disrupt the Raf2-Cul4 interaction (Figure 5C). Further Y2H assays indicate that neither the RFTS domain nor the zinc finger domain of Raf2 alone were sufficient to mediate the interaction with Cul4 (Figure 5B). These data demonstrate that the RFTS domain is necessary, but not sufficient, for integrating Raf2 within the CLRC complex suggesting that the overall tertiary structure of full-length Raf2 may be important to mediate the Raf2-Cul4 interaction (Figure 5B).siRNA production is maintained in RFTS mutantsThe CLRC complex is required for siRNA production [13,21]. However, it has been shown that specific point mutations in the CLRC components Raf1 and Cul4 cause disruption of heterochromatin whilst siRNA generation is unaffected [23,48]. To determine whether siRNA generation is disrupted by mutations within the RFTS domain northern analysis was performed.PMID:23357584 We observed that, unlike dcr1D and raf2D mutants, centromeric siRNAs are produced at wild-type levels in raf2-I98A, raf2-S100F and raf2-E104 cells at both 25uC and 36uC (Figure 4). Therefore, it appears that, as reported for specific raf1 and cul4 mutants, point mutants within the RFTS domain of Raf2 uncouple siRNA production from H3K9 methylation. These analyses provide additional support for the finding that CLRC is absolutely required for H3K9 methylation atPLOS ONE | www.plosone.orgThe RFTS Domain of Raf2 Is Required for Heterochromatin IntegrityFigure 6. Schematic diagram of heterochromatin defect in Raf2 RFTS mutants. Cells containing point mutations within the RFTS domain of Raf2 maintain an intact CLRC at 25uC, siRNAs are generated from non-coding RNA transcripts originating from the centromere and chromat.
Posted inUncategorized