| 2007 |
SLIP1 (MIF4GD) was identified as a binding partner of SLBP via yeast two-hybrid screening. Five conserved residues in a 15-amino-acid region of SLBP's N-terminal portion are required for both translation activation and SLIP1 binding; mutations in any of these residues reduce SLIP1 binding. Co-expression of SLIP1 with human SLBP in Xenopus oocytes stimulates translation of a stem-loop reporter mRNA but not a polyadenylated reporter, and siRNA-mediated knockdown of SLIP1 in HeLa cells reduces histone mRNA translation rate and cell viability. |
Yeast two-hybrid screen, site-directed mutagenesis, Xenopus oocyte translation assay, RNAi knockdown in HeLa cells, GFP reporter assay |
Molecular and cellular biology |
High |
18025107
|
| 2013 |
Crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP was solved at 2.5 Å resolution, defining the molecular determinants of SLBP recognition. A SLIP1-binding motif (SBM) was identified in two additional proteins, eIF3g and the mRNA-export factor DBP5; the 3.25 Å crystal structure of SLIP1 bound to the DBP5 SBM was also determined. Pull-down assays confirmed SLIP1 binding to DBP5 and eIF3g. SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CTIF, suggesting SLIP1 homodimer or SLIP1-CTIF heterodimer can bridge SLBP with SBM-containing proteins in mRNA metabolism. |
X-ray crystallography (2.5 Å and 3.25 Å resolution structures), pull-down assays, sequence conservation analysis |
Nucleic acids research |
High |
23804756
|
| 2013 |
SLIP1 is a homodimer that does not bind RNA on its own. Unphosphorylated SLBP-SLIP1 forms a 2:2 high-affinity (Kd <0.9 nM) heterotetramer incapable of binding histone mRNA. Phosphorylated SLBP (phosphorylated at 23 Ser/Thr sites) has weak affinity (~3 µM) for SLIP1. Sequential binding — phosphorylated SLBP to the histone mRNA stem-loop, then SLIP1 — is required to form an active ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to a heterodimer. Alanine scanning mutagenesis mapped the SLBP-binding site on SLIP1 to near the dimer interface; a single-point mutant there abolished SLBP interaction in vitro and reduced histone mRNA abundance in vivo. |
Biophysical characterization (sedimentation, binding assays), baculovirus phosphorylated SLBP expression, alanine scanning mutagenesis, in vivo histone mRNA measurement |
Biochemistry |
High |
23286197
|
| 2012 |
INT6/EIF3E (an eIF3 subunit) was identified as a binding partner of MIF4GD/SLIP1 via yeast two-hybrid screen. INT6 and MIF4GD co-localize in cytoplasmic foci. siRNA knockdown of INT6 reduces S-phase histone mRNA translation, assessed by endogenous histone protein levels and luciferase reporter driven by histone stem-loop elements; INT6 overexpression has the opposite effect. INT6 interacts with both MIF4GD and SLBP, placing it in the histone mRNA translation complex. |
Yeast two-hybrid screen, RNAi knockdown, luciferase reporter assay, co-localization by immunofluorescence |
RNA (New York, N.Y.) |
Medium |
22532700
|
| 2013 |
MIF4GD was identified as a novel binding partner for the CDK inhibitor p27(kip1) via yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation and GST pull-down assays. MIF4GD interaction with p27 stabilizes p27 in both the nucleus and cytoplasm by suppressing CDK2-mediated phosphorylation of p27 at Thr187. MIF4GD overexpression increases p27 levels and reduces cell proliferation; MIF4GD knockdown promotes cell cycle progression with decreased p27 levels. MIF4GD overexpression reduces colony formation and inhibits xenograft tumor growth in nude mice. |
Yeast two-hybrid screen, co-immunoprecipitation, GST pull-down, overexpression and knockdown in hepatocellular carcinoma cells, xenograft tumor model |
Oncogene |
Medium |
24336329
|
| 2010 |
MIF4GD/SLIP1 was identified as a candidate binding partner of supervillin (via yeast two-hybrid screen); the MIF4GD-interacting sequence co-localizes with and mis-localizes EGFP-supervillin in mammalian cells, and MIF4GD-interacting sequences mimic supervillin overexpression by inhibiting cell spreading. |
Yeast two-hybrid screen, co-localization of EGFP-supervillin with interacting sequences in mammalian cells, cell spreading assay |
Cytoskeleton (Hoboken, N.J.) |
Low |
20309963
|
| 2010 |
In rat testis, MIF4GD exists in three isoforms (25, 20, and 16 kDa). The 20-kDa form is testis-specific. The 16-kDa MIF4GD uniquely co-fractionates with 40S ribosomal subunits and ribosomes in spermatogenic cells by subcellular fractionation, suggesting it functions as a translational regulator in spermiogenesis. |
Western blot, subcellular fractionation (ribosome sedimentation), tissue expression analysis |
The Journal of reproduction and development |
Low |
21157122
|