Fig. 5. Mutations in mouse coilin affect nuclear body formation. (A) Alignment of the C-termini of human, mouse and frog coilin. The HindIII site used in generating the coilin chimeras is shown. (B) Summary of the mutations generated in this study. All constructs are fused to the C-terminus of GFP, with the exception of human coilin-GFP (FL-GFP). The constructs were transfected into MEF cells lacking endogenous coilin (Tucker et al., 2001) and scored for their ability to generate foci. Note that human constructs coilin-GFP and (1-481) as well as mouse construct (1-477) produce numerous unregulated foci. (C) HeLa cells were transfected with GFP-tagged wild-type mouse coilin or mutations of mouse coilin. Endogenous coilin (right column) was detected by using an antibody specific to human coilin (R508). Arrows mark the transfected cells, with concurrent disruption of Cajal bodies (right panels). Arrowheads show non-transfected cells, which display normal coilin localization in CBs and the nucleoplasm (right column). Foci formed by GFP-mouse coilin-SST to DDT were able to recruit SMN and fibrillarin, whereas loss of coilin foci upon overexpression of GFP-mouse coilin-SST to AGA also correlated with a loss of SMN foci (data not shown).