In metazoans, the majority of histone proteins are generated from replication-dependent histone mRNAs. These mRNAs are unique in that they are not polyadenylated but have a stem-loop structure in their 3′ untranslated region. An early event in 3′ end formation of histone mRNAs is the binding of Stem-Loop Binding Protein (SLBP) to the stem-loop. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RBD for the histone mRNA hairpin. The first binding site (Glu129-Val158) consists of a helix-turn-helix (HTH) motif that likely recognizes the unpaired uridines in the loop of the histone hairpin and upon binding, destabilizes the first G-C base-pair at the base of the stem. The second binding site lies between residues Arg180-Pro200 which appears to recognize the second G-C basepair from the base of the stem and possibly regions flanking the stem-loop. We show that the SLBP-histone mRNA complex is regulated by threonine phosphorylation and proline isomerization in a conserved TPNK sequence that lies between the two binding sites. Threonine phosphorylation increases the affinity of SLBP for histone mRNA by slowing the off-rate for complex dissociation whereas the adjacent proline acts as a critical hinge that may orient the second binding site for formation of a stable SLBP-histone mRNA complex. The NMR and kinetic studies presented here provide a framework for understanding how SLBP recognizes histone mRNA and highlight possible structural roles of phosphorylation and proline isomerization in RNA-binding proteins in remodeling ribonucleoprotein complexes.

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 (KD < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has weak affinity (KD ~3 µM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop, 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 histone mRNA abundance 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 hetero-tetramer, or alternatively, formation of the SLIP1-SLBP hetero-tetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

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