FIGURE 5.

Y14 promotes formation of larger PRMT5-containing complex. A, cytoplasmic extracts prepared from mock, FLAG-Y14-, or DDX3-expressing stable HEK293 cells were fractionated on a 5–20% sucrose gradient. Odd-numbered fractions were analyzed by immunoblotting with antibodies against PRMT5, pICln, transportin (TRN), or FLAG epitope. B, pooled 25 S fractions from mock cell extract (lanes 1 and 3) or FLAG-Y14-expressing extract (lanes 2 and 4) were subjected to immunoprecipitation (IP) by using Y12 antibody. Total proteins (lanes 1 and 2) and Y12 co-precipitates (lanes 3 and 4) were immunoblotted using anti-PRMT5 and anti-SNRPB. C, as in B, the 25 S fractions were subjected to cross-linking in the absence (−) or presence (+) of glutaraldehyde (GA) followed by immunoblotting using anti-PRMT5. D, in vitro methylation of GST-SmD1 was performed in the 25 S fractions using ³H-labeled methyl donor. GST-SmD1 was recovered and shown by autoradiography (Auto-radio.) (upper panel) and Coomassie (Cooma.) Blue staining (lower panel). Mo, monomer; Mu, multimer; Di, dimer.