FIGURE 6.

The coiled-coil domain of NSrp70 influences oligomerization and alternative splicing but not binding to mRNA. A, Schematic of wild-type and coiled-coil region point mutants of NSrp70 (left panel) and analysis of the predicted probability of oligomerization through the coiled-coil domain using the COILS program (right panel). M, molecular mass; NLS, nuclear localization signal; CCD, coiled-coil deleted NSrp70. B, HEK293T cells were transiently transfected with Myc_NSrp70 and its mutants. The cross-linking assay was performed as described in Fig. 5B. The ratio of oligomerization of NSrp70 mutants is shown as a bar graph (right panel). Data are representative of three independent experiments. M, monomer; T, trimer; D, dimer; GA, glutaraldehyde. C and D, HEK293T cells were cotransfected with Myc_NSrp70 constructs and the CD44 minigene. C, the splicing activity and protein expression were determined as described in Fig. 1A. Data are representative of three independent experiments. EV, pCS4–3Myc; Relative in/ex ratio, relative inclusion band/exclusion band ratio; IB, immunoblotting. *, p < 0.05 versus pCS4–3Myc. D, after cell lysis, the sample was immunoprecipitated with the indicated antibodies (anti-mouse IgG or anti-Myc). RNAs purified from the immunoprecipitated samples and whole lysates were determined by RT-PCR. Expression of NSrp70 and its mutants was confirmed by Western blotting with the indicated antibodies.