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.</p

Yeast two-hybrid screening identifies hyaluronan/mRNA-binding protein family members: SERBP1 and HABP4 as binding proteins of SPIN1.

<p>(A) Yeast cells transformed with different plasmids were grown on selective medium under permissive condition (24°C) and restrictive condition (37°C). i and ii are positive controls, iv and vi are negative controls, iii and v are yeast cells with positive clones from the yeast two-hybrid screening, co-transformed with bait plasmid containing <i>Spin1</i>. (B) Summary of the yeast two-hybrid screening. (C) HA-tagged SERBP1 is co-immunoprecipitated with MYC-tagged SPIN1 using MYC-antibody and extract from HEK293T cells expressing HA-SERBP1 and MYC-SPIN1. (D) HA-tagged HABP4 is co-immunoprecipitated with MYC-SPIN1 using MYC-antibody and extract from HEK293T cells expressing HA-HABP4 and MYC-SPIN1.</p

Summary of the genetic crosses of Spin1-GT heterozygous.

<p>Based on analyses of 19 litters from 6 mating pairs, ∼4 pups/litter.</p

<i>Spin1</i> mutant undergoes normal folliculogenesis and oocyte growth, but exhibit defective meiotic resumption.

<p>(A) Haematoxylin and eosin staining of ovarian sections grafted under the kidney capsules. Left panel: wild type; right panel: <i>Spin1</i> mutant. Scale bar represents 500µm. (B) Fully grown oocytes harvested from wild type and <i>Spin1</i> mutant ovaries after stimulation by PMSG. Scale bar represents 50µm. (C) Immunofluorescence staining of fully grown oocytes by anti-SPIN1 (green) and anti-Tubulin (red) antibodies. DNA (blue) was visualized by Hoechst 33342 dye. Scale bar represents 50µm. (D) Quantification of the percentage of fully grown oocytes with GV after release from the phosphodiesterase inhibitor IBMX. Data are mean ± SEM, Student’s t-tests.</p

Maternal <i>Pde3A</i> mRNA is reduced in the <i>Spin1</i> mutant oocytes.

<p>Expression levels of <i>Spin1</i>, <i>Serpine1</i>, <i>Adcy3</i>, <i>Pde3A</i>, and <i>SpdyA</i> were measured by real-time quantitative PCR (qPCR) using mRNAs isolated from wild type- and <i>Spin1</i>-mutant fully grown oocytes, then reversed transcribed to cDNA. The cDNAs were pre-amplified prior to qPCR. Data are mean ± SEM, Student’s t-tests.</p