Binding and methylation activities of recombinant PRMT5 methylosome. (A) PRMT5/MEP50 complex without (lane 1) or with pICln (lane 2) bound to Lsm10/11 heterodimer was purified on amylose beads via MBP attached to Lsm10 and analyzed by silver staining. (B) Lsm10/11ΔNL heterodimer was tested for the ability to stably bind PRMT5/MEP50 complex either in the absence (lane 1) or in the presence of pICln (lane 2). Proteins purified on amylose beads via MBP attached to Lsm10 were visualized by silver staining. Lanes 3–4 represent 5% input of proteins used in the binding assay. (C) Various Sm or Lsm heterodimers were incubated overnight in solution with recombinant complex of PRMT5 and MEP50 in the presence of 3H SAM. Proteins were resolved by SDS-PAGE and visualized by staining with Coomassie Blue (lanes 1–7). Dried gel was used for fluorography to detect proteins labeled with radioactive methyl group (lanes 8–14). (D) Various amino-terminal fragments of Lsm11 fused at the amino terminus with MBP were tested for their ability to undergo methylation, as described in panel C. Coomassie Blue stained gel and fluorogram are shown at the top and bottom, respectively. Asterisks in the top panel indicate full-length amino-terminal Lsm11 fragments fused to MBP. Lower bands correspond to proteolytic fragments.
