Amino acid conservation) for each alignment column: red (greater than 3), violet (between 3 and 1.5) and light yellow (between 1.5 and 0.5). Sequences are named according to their UniProt names. Full species names are: RAF2_SCHPO, Schizosaccharomyces pombe; S9X856_SCHCR, Schizosaccharomyces cryophilus; B6K1K8_SCHJY, Schizosaccharomyces japonicus; L8FMY4_PSED2, Pseudogymnoascus destructans; G2Q7S7_THIHA, Thielavia heterothallica; B2B7F9_PODAN, Podospora anserina; G4ML14_MAGO7, Magnaporthe oryzae; C1GEE7_PARBD, Paracoccidioides brasiliensis; J3K2M4_COCIM, Coccidioides immitis; A2Q7V8_ASPNC, Aspergillus niger; D4DHS5_TRIVH, Trichophyton verrucosum; K2RLQ8_MACPH, Macrophomina phaseolina. Residues which are subject to mutation are labeled. RFTS and C2H2 Zinc Finger domains are boxed in violet and red, respectively. B. Raf2 protein missing the entire RFTS domain does not encode a truncated protein. C. Zoom-in of RFTS structure showing the region containing the point mutations. (TIF)Figure S1 Figure S2 A. Western blot demonstrating that both wild typeThe Raf2 RFTS domain is required for heterochromatin integrity but not siRNA generationCLRC has two major functions in heterochromatin formation: it possesses histone methyltransferase activity via Clr4 and mediates siRNA production [21,51]. In wild-type fission yeast, these processes are coupled to direct heterochromatin formation to specific location such as centromeres, telomeres and the silent mating-type locus, and prohibit silencing elsewhere (Figure 6). Cells expressing only mutant histone H3 (H3K9R) are unable to methylate K9 of H3 and do not form heterochromatin, however such cells continue to produce a low level of siRNAs homologous to centromeric repeats [52,53].Amifostine This suggests that the CLRC complex plays a role in promoting siRNA production, independently of H3K9 methylation.Zalcitabine Deletion of any CLRC component results in loss of both H3K9 methylation and siRNA production, yet point mutations within CLRC components Raf1 and Cul4 exhibit separable functions with respect to chromatin modification and siRNA generation [23,48].PMID:24576999 We demonstrate here that specific mutations within the RFTS domain of Raf2 result in the loss of the classic marks of heterochromatin, namely H3K9 methylation and Swi6, but maintain siRNA production. Thus, as previously documented for specific mutations within Raf1 and Cul4, mutation in the RFTS domain of Raf2 uncouple chromatin modification from siRNA production [23,48]. This effect may be due to partial disruption of the CLRC; the point mutants studied may be able to maintain specific interactions required for siRNA generation but lose those that are critical for H3K9 methylation and subsequent protein associations. It may be that, as seen in specific Raf1 mutants, siRNA levels remain high because the defective Raf2 RFTS mutations inhibit the degradation of pre-existing siRNAs [23]. Another tenable explanation is that the particular Raf2 RFTS mutants analysed do not disrupt the continual synthesis of siRNAs from centromere repeat transcripts. In fact, since Raf2 has been shown to interact with Cdc20, this could provide a molecular link between DNA replication, siRNA production and chromatin modification [25]. More extensive analyses of such interactions in cells harboring mutations such as those in the Raf2 RFTS mutation should provide insight into the interplay between Raf2, Cdc20 and the role of DNA replication in these processes andand proteins containing point muta.
