Interaction of the Histone
mRNA Hairpin with Stem–Loop Binding Protein (SLBP) and Regulation
of the SLBP–RNA Complex by Phosphorylation and Proline Isomerization
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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