Abstract
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 structure. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RNA binding domain for the histone mRNA hairpin. The first binding site (Glu129-Val158) consists of a helix-turn-helix 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 and Pro200, which appears to recognize the second G-C base pair from the base of the stem and possibly regions flanking the stem-loop structure. 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 nuclear magnetic resonance 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.