Figure 6.

Both FKBDs and the TPR domain of Coi12p are important for scnRNA loading in vivo

1. A schematic drawing of the in vivo functional analysis of COI12 mutants. The expression constructs from which HA-tagged Coi12p proteins (wild-type [WT] or the mutant proteins lacking the two FKBDs [ΔFKBD] or the TPR domain [ΔTPR]) are expressed under the control of the cadmium-inducible MTT1 promoter were introduced into the MACs of COI12KO cells.
2. Cells containing the expression constructs shown in (A) were mated with COI12KO cells in the presence of cadmium ions. The localization of Twi1p was analyzed by immunofluorescence staining using an anti-Twi1p antibody at 3 hpm (B–D). In (B–D), the parental MACs are marked with arrowheads. DNA elimination was analyzed by DNA FISH with probes against Tlr1 IESs at 36 hpm (E). Defects in DNA elimination were categorized as described for Fig1C. The expression of the different Coi12p mutants was analyzed by Western blot using an anti-HA antibody (F, top). Alpha-tubulin was analyzed as a loading control (F, bottom). The Twi1p-containing complex was immunoprecipitated with an anti-Twi1p antibody. Precipitated scnRNA was separated on a denaturing gel and visualized by the nucleic acid staining dye GelRed (G, top and middle). The intensities of bands relative to the sample with the mating expressing wild-type Coi12p (KO+WT) are shown on the top, and a result of a representative experiment is shown in the middle. Precipitated Twi1p was analyzed by Western blot using an anti-Twi1p antibody (bottom). In (G), the standard deviation (SD) between technical replicates is indicated.