Lsm11 interacts with the PRMT5 methylosome. (A) Immunoprecipitation of HeLa (lanes 1–3) or mouse proteins (lanes 4–7) bound to recombinant heterodimers of Lsm10 with either wild-type Lsm11 (labeled as 10/11), Lsm11 lacking the internal loop (10/11ΔL), or Lsm11 lacking both the amino-terminal fragment and the loop (10/11ΔNL). The heterodimers were incubated with indicated extracts and precipitated with the α10/11 rabbit serum. Immunocomplexes were collected on protein A beads, resolved by SDS-PAGE and protein bands stained with silver. Proteins precipitated by the same serum from HeLa or mouse extracts lacking recombinant Lsm10/11 heterodimer are shown in lanes 1,6. HC and LC denote heavy and light chains of immunoglobulins, respectively. (B) Sequence of human Lsm11 (amino acid 1–360). The boundaries of the deleted regions are indicated with arrowheads. Arginine residues neighboring glycines potentially targeted for methylation by PRMT5 are indicated with arrows. Structural elements within the Sm fold are indicated with a horizontal bar (α-helix) or thick arrows (β-stands). The portion of the internal loop between β-stands 3 and 4 that was deleted in Lsm11ΔL and Lsm11ΔNL is underlined. (C–E) Purification of PRMT5 methylosome on amylose beads via maltose-binding protein (MBP) tag attached to Lsm10 or to the amino-terminal fragments of Lsm11. HeLa or mouse cytoplasmic extracts, as indicated, were incubated with variants of Lsm10/11 heterodimers or amino-terminal fragments of Lsm11 fused to MBP, and proteins collected on amylose beads were analyzed by silver staining (panels C and E) or western blotting, using specific antibodies (panel D). In panel E, asterisks indicate full-length amino-terminal Lsm11 fragments fused to MBP.
