Tudor domains are required for SPIN1 functions.

<p>(A) Structural alignment of three SPIN1 Tudor domains with the SMN Tudor domain, adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069764#pone.0069764-Zhao1" target="_blank">[15]</a>. Amino acid residues important for the structural fold of Tudor domain are highlighted in green. The glutamate residue involved in SMN protein-protein interactions is denoted by an asterisk. (B) Three-dimensional structure of human SPIN1 (PDB ID: 2NS2). The three tyrosine residues (Y98, Y177, Y254) of human SPIN1 are displayed. (C) Co-immunoprecipitation assay of SERBP1 with SPIN1 containing various mutations in the Tudor domains. HEK293T cells were transfected with MYC-tagged SPIN1 wild type or with the MYC-tagged SPIN1 point-mutants. The protein complexes were pulled down with MYC-antibody, and the endogenous SERBP1 was probed using SERBP1-antibody. (D) Luciferase reporter assay to measure the RNA-binding activity of SERBP1. HEK293T cells were transfected with luciferase reporters carrying the Serpine1 3′UTR, and were then co-transfected with the various plasmids. The RNA-binding activity of the endogenous SERBP1 was measured based on the relative expression of luciferase. Data are mean ± SEM, Student’s t-tests.