Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.

Nitin Bansal, Minyou Zhang, Aishwarya Bhaskar, Patrick Itotia, EunHee Lee, Lyudmila S Shlyakhtenko, TuKiet T Lam, Andrew Fritz, Ronald Berezney, Yuri L Lyubchenko, Walter F Stafford, Roopa Thapar

Biochemistry 2013 Jan 22

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The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high-affinity (K(D) < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (K(D) ~3 μM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop motif followed by association with SLIP1 is required to form an "active" ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1-SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

Citation

Nitin Bansal, Minyou Zhang, Aishwarya Bhaskar, Patrick Itotia, EunHee Lee, Lyudmila S Shlyakhtenko, TuKiet T Lam, Andrew Fritz, Ronald Berezney, Yuri L Lyubchenko, Walter F Stafford, Roopa Thapar. Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1. Biochemistry. 2013 Jan 22;52(3):520-36