Coilin Methylation Regulates Nuclear Body Formation Michael D. Hebert, Karl B. Shpargel, Jason K. Ospina, Karen E. Tucker, A.Gregory Matera  Developmental Cell  Volume 3, Issue 3, Pages 329-337 (September 2002) DOI: 10.1016/S1534-5807(02)00222-8

Figure 1 Schematic of the Mouse Coilin RG Box and Five Mutants Used in the SMN Recruitment Assay Consensus sites for symmetrical dimethylation are underlined. The R to K mutation represents substitution of the indicated arginines to lysines in an otherwise intact RG box. The other four constructs [coilinΔRG+(RG)4, coilinΔRG+(RA)4, coilinΔRG+(KG)4, and coilinΔRG+(AG)4] were generated by mutagenesis of a preexisting RG box deletion plasmid (coilinΔRG). These constructs (all GFP tagged) were cotransfected with myc-SMN into mouse embryonic fibroblast cells lacking endogenous coilin and assayed for their ability to incorporate SMN into CBs. It is important to note that cotransfection with SMN is not required for this assay, as the transfected coilin does recruit endogenous SMN protein (see Supplemental Figure S1 at http://www.developmentalcell.com/cgi/content/full/3/3/329/DC1). However, the coilin knockout MEFs do not often display gems (Tucker et al., 2001). Thus, to efficiently score large numbers of cells that are not only transfected but also contain SMN foci, myc-tagged SMN was cotransfected along with the appropriate coilin construct (Hebert et al., 2001). Example images showing recruitment of SMN into CBs (left panel, yellow dot) or its exclusion in gems (right panel, red dots) are shown. Note that separate green coilin foci are also observed (right panel). Developmental Cell 2002 3, 329-337DOI: (10.1016/S1534-5807(02)00222-8)

Figure 2 The Interaction between Coilin and SMN In Vivo Is Modulated by Methylation (A) HeLa cells were transfected with a myc-tagged coilin fragment [myc-coilin(ΔNΔC)]. Where indicated, the methyltransferase inhibitor Adox was added and the cell lysates were immunoprecipitated with anti-myc antibodies. The blot was probed with antibodies to SMN (top panels), and then the same blot was reprobed with anti-myc antibodies (bottom panels). The input lanes reflect 8% of the lysate used in the IPs. (B) The canonical anti-Sm antibody Y12 recognizes coilin in a methylation-dependent fashion. HeLa cells were either mock transfected or transfected with GFP-coilin, with or without the addition of Adox. Following immunoprecipitation with anti-GFP, the samples were analyzed by Western blot with the anti-sDMA antibody Y12 (top panel). The bottom panel shows the same membrane reprobed with anti-GFP to confirm the location and quantity of immunoprecipitated GFP-coilin. Developmental Cell 2002 3, 329-337DOI: (10.1016/S1534-5807(02)00222-8)

Figure 3 The Anti-sDMA Antibody Y12 Can Recognize Coilin In Vivo MEF cells lacking endogenous coilin were transfected with coilin fused to the amino terminus of GFP (coilin-GFP). The presence of snRNPs in these foci was ascertained by immunofluorescence or RNA FISH. The GFP signal for each cell is shown in the left panels, and the various snRNP markers are shown on the right. Arrows indicate example foci that are reactive to anti-Sm (Y12), but not positive for U2B″, TMG-cap, or U7 snRNA. Developmental Cell 2002 3, 329-337DOI: (10.1016/S1534-5807(02)00222-8)

Figure 4 SMN Interaction with Coilin Is Reduced in the Methylation-Deficient HeLa-PV Cell Line (A) Coilin/SMN interaction is reduced in HeLa-PV in vivo compared to that in HeLa-ATCC. GFP-coilin was transfected into HeLa-ATCC cells, with or without Adox, as well as in HeLa-PV cells. This was followed by immunoprecipitation with anti-GFP. Western analysis with the anti-sDMA mAb Y12 (top panels) revealed the extent of coilin dimethylation. Note that GFP-coilin can be detected in the anti-sDMA input lanes (1–3) in an Adox-sensitive manner. Subsequently, the blot was reprobed with anti-SMN mAb 7B10 (middle panels) and anticoilin Ab R288 (bottom panels). The input lanes represent 8% of the amount used in the IPs. (B) HeLa-PV extract undermethylates both coilin and SmD3 constructs in vitro. The C-terminal RG-rich regions of both coilin and SmD3, fused to GST, were used as substrates in an in vitro SMN binding/methylation assay. In the upper panels, equimolar amounts of GST-C214 or GST-SmD3 tail were incubated with extract from HeLa-ATCC or HeLa-PV cells, with or without the addition of Adox (5 mM final concentration) for 1 hr at 30°C. The samples were washed and analyzed by Western blotting with anti-SMN. The input lanes account for 5% of the extract used in the reactions. In the lower panels, the activities of HeLa-ATCC, -PV, and -KN cells were compared with the same SMN binding assay. HeLa-KN cells show a similar CB-gem separation phenotype to that of HeLa-PV. (C) To demonstrate that coilin is a methylation substrate, HeLa-ATCC extract was incubated with either GST-C214 or GST-SmD3 tail in the presence of 3H-SAM (left panels). Symmetrical dimethylation in both HeLa-ATCC and -PV extracts (right panels) was assayed by immunoprecipitation of His-tagged coilin and SmB′ proteins with anti-sDMA mAb Y12 (see Methods). Radiolabeled proteins were detected by fluorography. Developmental Cell 2002 3, 329-337DOI: (10.1016/S1534-5807(02)00222-8)

Figure 5 Cajal Bodies Are Enriched in U snRNPs in the Absence of SMN HeLa-PV cells were grown at 32°C to exacerbate the separation phenotype and then assayed for colocalization of coilin, SMN, and U snRNPs with Ab R508, mAb 2B1, and antisense in situ oligoprobes, respectively. U2 (top) and U7 (bottom) snRNAs were visualized by RNA FISH. In cells that display both gems and CBs, U snRNPs were enriched in the Cajal bodies lacking SMN. Arrows in the U2, U7, and coilin panels indicate the presence of U snRNPs in CBs. Arrows in the SMN panels indicate positions of the CBs that lack SMN, which is enriched in the cytoplasm and gems. Developmental Cell 2002 3, 329-337DOI: (10.1016/S1534-5807(02)00222-8)