A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies

Abstract

SummaryThe spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2-8 (Gemin2-8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1c. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1c to CBs. Moreover, depletion of PP1c by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1c-depleted cells. Twodimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1c-depleted cells and expression of PP1c restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1c in the atrophic skeletal muscles, suggesting that the function of PP1c is likely to be affected in disease. Our findings reveal a role of PP1c in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1c as a target for therapeutic intervention in SMA.

Signals controlling Cajal body assembly and function

“…The majority of coilin, for example, is nucleoplasmic. Shown above the CB are enzymes known or suspected to modify coilin or other components of the CB: PPM1G (protein phosphatase, Mg 2+ /Mn 2+ dependent, 1G) (Hearst et al, 2009, Petri, Grimmler, 2007), CK1/2 (casein kinase 1 (our unpublished results) and 2) (Hebert and Matera, 2000), VRK1/2 (vaccinia-related kinase 1 and 2) (Sanz-Garcia et al, 2011), PRMT5/7 (protein arginine methyltrasferase 5 and 7) (Boisvert, Cote, 2002, Gonsalvez, Tian, 2007), protein phosphatases PP2A and PP1(γ), with adaptor subunit p99 (PNUTS) (Kitao et al, 2008, Lyon et al, 1997, Moorhead et al, 2007, Renvoise et al, 2012) and Cdk2/cyclinE (Liu et al, 2000). The ubiquitin-specific protease-like 1 (USPL1) SUMO isopeptidase protein has been shown to impact coilin localization (Schulz, Chachami, 2012).…”

SMN - A chaperone for nuclear RNP social occasions?

“…[91][92][93][94][95] In addition to overall protein levels, post-translational modifications (PTMs) of resident proteins, including SMN and coilin, are almost certainly important for establishing and maintaining inter-and intra-molecular interactions within Cajal bodies. [96][97][98][99][100] Disruption of these PTMs can result in mislocalization of SMN and alterations in Cajal body integrity. These and other factors will need to be addressed by future studies.…”

The tudor protein survival motor neuron (SMN) is a chromatin-binding protein that interacts with methylated histone H3 lysine 79

“…The SMN Tudor domain binds preferentially to symmetric dimethyl-arginine (sDMA) residues in Arg-Gly (RG)-rich sequences of spliceosomeassociated Sm family proteins and in coilin, the major structural component of the nuclear domains known as Cajal bodies (CBs) (Boisvert et al, 2002;Côté and Richard, 2005;Friesen et al, 2001;Liu et al, 2012;Raska et al, 1991;Selenko et al, 2001;Tripsianes et al, 2011). The localization of SMN to CBs depends on its interaction with methylated and phosphorylated coilin (Hearst et al, 2009;Hebert et al, 2002;Hebert et al, 2001;Toyota et al, 2010) and phosphorylation of SMN itself (Petri et al, 2007;Renvoisé et al, 2012). Following hypomethylation of coilin, SMN complexes form individual nuclear bodies called Gemini bodies or gems juxtaposed to CBs (Liu and Dreyfuss, 1996).…”