Coilin Methylation Regulates Nuclear Body Formation

Abstract

functional homolog, yeast Rmt1/Hmt1, encoding the bulk of the activity (Tang et al., 2000; McBride et al., Center for Human Genetics and Program in Cell Biology 2000). The only type II PRMT defined to date is the Janus kinase binding protein, JBP1/PRMT5 (Branscombe et Case Western Reserve University and University Hospitals of Cleveland al., 2001). There are only five known targets of PRMT5: myelin Cleveland, Ohio 44106 basic protein (Baldwin and Carnegie, 1971) and the spliceosomal proteins SmB/BЈ, SmD1, SmD3, and Lsm4 (Brahms et al., 2000, 2001). Importantly, symmetrical Summary dimethylation of the Sm and Sm-like proteins is important for binding to the survival of motor neurons (SMN) Cajal bodies (CBs) are nuclear suborganelles involved in biogenesis of small RNAs. Twin structures, called protein in vivo (Friesen et al., 2001a; Brahms et al., 2001). The SMN1 gene is mutated in Ͼ95% of patients with gems, contain high concentrations of the survival motor neurons (SMN) protein complex. CBs and gems spinal muscular atrophy (SMA), a recessive neurodegenerative disease, which is the leading genetic cause of often colocalize, and communication between these subdomains is mediated by coilin, the CB marker. Coi-childhood mortality (Pearn, 1980). SMN is part of a large protein complex that is required for assembly of Sm lin contains symmetrical dimethylarginines that modulate its affinity for SMN, and, thus, localization of SMN proteins onto spliceosomal U snRNAs (Liu et al., 1997; Fischer et al., 1997; Meister et al., 2000, 2001a). The complexes to CBs. Inhibition of methylation or mutation of the coilin RG box dramatically decreases bind-protein is localized diffusely throughout the cytoplasm as well as to nuclear Cajal bodies (Liu and Dreyfuss, ing of coilin to SMN, resulting in gem formation. Coilin is hypomethylated in cells that display gems, but not 1996; Matera and Frey, 1998; Carvalho et al., 1999). We have recently shown that coilin, the Cajal body (formerly in those that primarily contain CBs. Likewise, extracts prepared from cells that display gems are less efficient coiled body) marker protein, interacts with SMN both genetically (Tucker et al., 2001) and physically (Hebert in methylating coilin and Sm constructs in vitro. These results demonstrate that alterations in protein methyl-et al., 2001). The interaction between coilin and SMN is direct, and recruitment of the SMN complex to Cajal ation status can affect nuclear organization. bodies (CBs) depends upon the presence of an arginineand glycine-rich domain (the RG box motif) within coilin

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

“…Therefore, purified Sm proteins from HeLa snRNPs (Raker et al 1996;Sumpter et al 1992) were used as a known source of sDMA-modified binding substrates. GST-pull-down assays demonstrated that SmB/B′, SmD3, and SmD1 were detected by monoclonal antibody Y12, which recognizes sDMA-modified substrates (Brahms et al 2001;Hebert et al 2002). We found that Sm proteins bound to the GST-coilin subfragment, but not to GST alone (Fig.…”

“…Symmetric dimethylarginine (sDMA) modification strongly increases the affinity of SMN for RG dipeptide repeats (Brahms et al 2001;Friesen et al 2001). Consistent with these observations, coilin hypomethylation results in delocalization of SMN to twin structures called Gemini of CBs, or gems (Boisvert et al 2002;Hebert et al 2002). In addition to SMN, coilin's direct binding partners include itself (Hebert and Matera 2000), the nucleolar protein Nopp140 (Isaac et al 1998) and SmB/B′ (Hebert et al 2001).…”

“…Since the CBs that are devoid of SMN maintain high concentrations of snRNPs (Liu and Dreyfuss 1996;Hebert et al 2002), SMN is unlikely to be this factor. Previously, we showed that the C-terminal 214 amino acid (aa) residues of coilin are sufficient for interaction with SmB′, making coilin a candidate (Hebert et al 2001).…”

“…Because the interaction of SMN with both coilin and Sm proteins was greatly enhanced by the presence of symmetrical dimethylarginine (sDMA) residues (Brahms et al 2001;Friesen et al 2001;Hebert et al 2002), we were interested to know whether coilin could bind to sDMAmodified Sm proteins. In the above experiments, the in vitro translated SmD1 (Fig.…”

“…The arginines within this so-called RG box motif are symmetrically dimethylated (Boisvert et al 2002;Hebert et al 2002). Symmetric dimethylarginine (sDMA) modification strongly increases the affinity of SMN for RG dipeptide repeats (Brahms et al 2001;Friesen et al 2001).…”