FIGURE 6.

Y14 associates with snRNP assembly complex. HEK293 cells were mock-transfected or transiently transfected with the expression vector FLAG-Y14 alone (B, D, and F) or together with the vector of HA-SmB (A). Anti-FLAG immunoprecipitates (IP) were subjected to immunodetection of tagged proteins (A) or endogenous proteins as indicted (D and F) or Northern blotting using antisense RNA probes complementary to U1, U2, U4, U5, and U6 (B). In B, subcellular fractionation was evaluated by immunoblotting using anti-PARP1 and anti-α-tubulin (lower panel). N and C represent nuclear and cytoplasmic fractions, respectively. C, the FLAG-REF and -Y14 expression vectors were transiently transfected into HEK293 cells. Proteins that co-precipitated with anti-trimethylguanosine were immunoblotted with anti-FLAG. In D, anti-Gemin3 cross-reacted to a protein band indicated by the asterisk. E, HEK293 cells were mock-transfected (lane 1) or were transfected with vector expressing FLAG-PRMT5 (lane 2), HA-Y14 alone (lane 4), or both protein-expressing vectors (lane 3). Immunoprecipitation was performed using anti-FLAG followed by immunoblotting using antibodies as indicated.