Abstract
pICln is an essential, highly conserved 26-kDa protein whose functions include binding to Sm proteins in the cytoplasm of human cells and mediating the ordered and regulated assembly of the cell's RNA-splicing machinery by the survival motor neurons complex. pICln also interacts with PRMT5, the enzyme responsible for generating symmetric dimethylarginine modifications on the carboxyl-terminal regions of three of the canonical Sm proteins. To better understand the role of pICln in these cellular processes, we have investigated the properties of pICln and pICln·Sm complexes and the effects that pICln has on the methyltransferase activity of PRMT5. We find that pICln is a monomer in solution, binds with high affinity (Kd ∼ 160 nm) to SmD3-SmB, and forms 1:1 complexes with Sm proteins and Sm protein subcomplexes. The data support an end-capping model of pICln binding that supports current views of how pICln prevents Sm oligomerization on illicit RNA substrates. We have found that by co-expression with pICln, recombinant PRMT5 can be produced in a soluble, active form. PRMT5 alone has promiscuous activity toward a variety of known substrates. In the presence of pICln, however, PRMT5 methylation of Sm proteins is stimulated, but methylation of histones is inhibited. We have also found that mutations in pICln that do not affect Sm protein binding can still have a profound effect on the methyltransferase activity of the PRMT5 complex. Together, the data provide insights into pICln function and represent an important starting point for biochemical analyses of PRMT5.
Highlights
PRMT5 has several known cellular targets, including myelin basic protein [6], histones [7], and the spliceosomal Sm proteins [8, 9]
When we looked at time courses for methylation of just the 32-residue carboxyl-terminal tail of SmD3 (D3C32), we found that pICln still stimulates PRMT5 methylation, to a lesser extent than for full-length SmD3 (Fig. 5B)
We have used the SmD3 protein and a heterodimeric complex containing SmD3 bound to the Sm domain of SmB as models to study the interaction of pICln with Sm proteins
Summary
Protein Expression and Purification—Proteins were overexpressed in BL21(DE3) cells at 37 °C for 3 h after induction with 0.5 mM isopropyl 1-thio--D-galactopyranoside. For full-length PRMT5 and the PRMT5 regulatory domain (residues 1–290), the GST fusions were co-expressed with pICln to promote the expression of soluble protein followed by affinity chromatography using glutathione-agarose beads (Sigma). For these co-ex- pICln Forms Tight 1:1 Complexes with Sm Proteins—Several pressions, GST fusions of pICln and pICln deletion mutants groups have shown that pICln interacts with spliceosomal Sm were expressed from pACYCDuet, and FLAG-tagged SmD3 proteins [8, 9, 21], yet the affinity and stoichiometry of these was expressed from pCDFDuet. Binding reactions each of the individual Sm proteins, with the exception of were washed, eluted with SDS, and visualized after SDS-PAGE SmE (supplemental Fig. 1) By employing these co-expresand PhosphorImager analysis of dried gels (GE Healthcare). By employing these co-expresand PhosphorImager analysis of dried gels (GE Healthcare). sion approaches, we have been able to produce soluble
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