Affinity chromatography with the RG domain of SmD3 isolates the methyltransferase JPB1. The indicated recombinant proteins (8 μg) were incubated with (+) or without (−) total HeLa extract. Following extensive washing, proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. The methyltransferase JBP1 (arrow) was identified by MALDI-TOF mass spectrometry, and pICln was identified by Western blot analysis (see Fig. 2). Numbers on the left are molecular masses in kilodaltons.

JBP1 and pICln associate with unmodified Sm proteins. (A) The indicated immobilized GST fusion proteins were incubated with HeLa cell extract, and bound proteins were analyzed by Western blotting to detect the indicated proteins (arrows). The total lane shows 10% of the extract used in each binding. (B) Streptavidin-immobilized biotin-linked peptides with (D1c29-sDMA and D3c32-sDMA) or without (D1c29 and D3c32) the specific sDMA modifications formed in vivo and biotin alone were incubated with HeLa cytoplasmic extract, and bound proteins were analyzed by Western blotting to detect the indicated proteins (arrows). The total lane shows 10% of the extract used in each binding. (C) Anti-SMN (2B1), anti-Sm protein (Y12), anti-pICln (α-pICln), and nonimmune (SP2/0) antibodies were used for immunoprecipitation of HeLa cytoplasmic extract, and bound cellular proteins were analyzed by Western blotting to detect the proteins indicated by arrows. The total lane shows 10% of the extract used in each immunoprecipitation.

The carboxyl-terminal RG domains of SmD1 and SmD3 are required for association with a 20S pICln-JBP1 complex. (A) 293 cell cytoplasmic extract was separated on a 5 to 20% sucrose gradient. Fractions (indicated by numbers) were collected, separated by SDS-PAGE, and Western blotted to detect the proteins indicated by arrows. (B) 293 cells transiently expressing myc-D3, myc-D3Δc32, myc-D1, or myc-D1Δc29 (as indicated) were separated on sucrose gradients, and fractions were collected, separated by SDS-PAGE, and immunoblotted to detect the myc-tagged proteins. The presence of the myc-tagged proteins did not affect the sucrose gradient migration pattern of pICln or JBP1 (data not shown). In both panels A and B, lanes P contain 5% of the pellet from the gradients. The total lanes contain 5% of the extract loaded on each gradient. (C) Fractions 2 to 5 and 12 to 15 (6S and 20S as indicated, respectively, in panel A) from the sucrose gradient separation of myc-D3- and myc-D3Δc32-expressing cytoplasmic extracts were pooled and immunoprecipitated with anti-Sm protein (Y12), anti-pICln (α-pICln), and nonimmune (SP2/0) antibodies as indicated. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted to detect the indicated proteins (arrows). The total lanes show 10% of the pooled fractions used in each immunoprecipitation.

Cytoplasmic SmD1/D2, SmD3, and SmB are associated with the 20S methylosome. Cytoplasmic extract was prepared from HeLa cells metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine and fractionated on a 5 to 20% sucrose gradient as for Fig. 3. Fractions 2 to 5 (6S) and 12 to 15 (20S) were pooled, immunoprecipitated with the anti-Sm (Y12), anti-pICln (α-pICln), and nonimmune (SP2/0) antibodies as indicated, and separated by SDS-PAGE. Radioactive signals were enhanced with Amplify (Amersham), and the gel was exposed to film. The Sm proteins SmB/B′, SmD3, SmD1/D2, SmE, SmF, and SmG, identified based on their molecular masses and presence in Y12 immunoprecipitates, are indicated. JBP1 was identified based its molecular mass and presence in the 20S anti-pICln immunoprecipitate. Similarly, pICln was identified based its size and presence in the 6S and 20S pICln antibody immunoprecipitates. Relatively minor unidentified proteins coimmunoprecipitated by anti-pICln from both the 6S and 20S regions of the gradient are indicated with black dots. Prominent proteins (p50 and p37) immunoprecipitated by anti-pICln in the 20S fraction are also indicated. The positions of molecular mass markers (in kilodaltons) are shown on the left.

pICln binds the Sm domains of SmD1 and SmD3. (A and B) The indicated in vitro-translated and [³⁵S]methionine-labeled proteins were incubated with GST or GST-pICln, and after washing, bound proteins were separated by SDS-PAGE and visualized by fluorography. (C) Binding of in vitro-translated pICln to the indicated immobilized proteins was performed as for panels A and B.

The methylosome. (A) Cytoplasmic extracts were prepared from 293 cells transiently expressing Flag-JBP1 and separated on a sucrose gradient. Fractions were collected, separated by SDS-PAGE, and Western blotted to detect Flag-JBP1, native JBP1, and pICln (arrows). (B) Fractions 12 to 15 (20S) were immunoprecipitated with anti-Flag beads, and the Flag peptide-eluted methylosome was separated by SDS-PAGE and Western blotted to detect Flag-JBP1, native JBP1, and pICln (arrows). (C) The purified methylosome was incubated with ³H-SAM and each protein substrate in 50 μl of binding buffer at 30°C for 30 min. The samples were then separated by SDS-PAGE, stained with Coomassie blue to check protein substrates, and exposed to film after treatment with Amplify to enhance radioactive signals.

JBP1 has methyltransferase specificity for SmD1, SmD3, and SmB. (A) Cytoplasmic extracts prepared from 293 cells transiently expressing Flag-JBP1, Flag-JBP1^R368A, or Flag alone (as indicated) were used for immunoprecipitation with immobilized anti-Flag antibody (α-flag IP). After elution with Flag peptide, a fraction of each immunoprecipitate was Western blotted with anti-JBP1 antibody. The total lanes contain 10% of the total extract used in each immunoprecipitation lane. (B) HeLa cytoplasmic extract was separated on a 5 to 20% sucrose gradient. Fractions 12 to 15 (20S) (see Fig. 3A) were pooled and immunoprecipitated with anti-pICln (α-pICln) and nonimmune (SP2/0) antibodies. A fraction of each immunoprecipitate was Western blotted with specific antibodies to detect JBP1 and pICln (arrows). The total lane contains 10% of the total extract blotted in each immunoprecipitation lane. (C to F) Flag-JBP1 (C), Flag-JBP1^R368A (D), or anti-pICln antibody immunoprecipitate from sucrose gradient fractions 12 to 15 (E) was incubated with ³H-SAM and each protein substrate in 50 μl of binding buffer at 30°C for 30 min. The samples were then separated by SDS-PAGE, stained with Coomassie blue, and treated with Amplify to enhance radioactive signals. (F) A representative Coomassie blue-stained gel showing the positions of the recombinant proteins. (G) His-tagged SmD1 and SmD3 were methylated with immunopurified Flag-JBP1, separated by SDS-PAGE, and visualized by fluorography as in panels C to F.

JBP1 is a type II protein arginine methyltransferase. GST-TEV-D3c32 was labeled with [³H]methyl groups by JBP1 or PRMT1 (as indicated). Following purification on glutathione-Sepharose beads and cleavage with TEV protease, the D3c32 peptides were hydrolyzed in acid; mixed with aDMA, sDMA, and MMA standard amino acids (as indicated); and separated on silica gel 60 thin-layer chromatography plates. The methylated arginine standards were visualized with ninhydrin, and ³H-methylated arginine residues were visualized on a phosphorimager.

SmD3 is transferred from the methylosome to the SMN complex. Cytoplasmic extract prepared from myc-D3-expressing 293 cells was separated on a sucrose gradient, and fractions 2 to 5 (6S) and 12 to 15 (20S) were pooled and incubated with SMN complex (FLAG-SMN) which had been purified on anti-Flag beads from cytoplasmic extract transiently expressing myc-SMN, Flag-Gemin2, myc-Gemin3, and myc-Gemin4 or with anti-Flag beads incubated with cytoplasmic extract prepared from vector-transfected cells (FLAG-vector) as indicated. After washing, retained proteins were analyzed by Western blotting to detect myc-D3 and JBP1 (arrows). The total lanes show 10% of the pooled fractions used in each binding.

Methylosome methylation of RG-containing Sm proteins targets them to the SMN complex for assembly into snRNP core particles. A schematic depicting Sm RG domain posttranslational methylation by the methylosome is shown. After sDMA modification, the Sm proteins associate with the SMN complex and, along with the other Sm proteins (which also bind SMN), are assembled on snRNA to form an snRNP core particle.