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Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.
- Source :
-
Biochemistry [Biochemistry] 2013 Jan 22; Vol. 52 (3), pp. 520-36. Date of Electronic Publication: 2013 Jan 11. - Publication Year :
- 2013
-
Abstract
- 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.
- Subjects :
- Carrier Proteins genetics
Histones chemistry
Histones genetics
Humans
Kinetics
Mutagenesis, Site-Directed
Mutant Proteins chemistry
Mutant Proteins metabolism
Nuclear Proteins genetics
Peptide Fragments chemistry
Peptide Fragments genetics
Peptide Fragments metabolism
Phosphorylation
Point Mutation
Protein Binding
Protein Interaction Domains and Motifs
Protein Multimerization
Protein Processing, Post-Translational
RNA Folding
RNA-Binding Proteins
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Serine chemistry
Serine metabolism
Threonine chemistry
Threonine metabolism
Tyrosine chemistry
Tyrosine metabolism
mRNA Cleavage and Polyadenylation Factors genetics
Carrier Proteins chemistry
Carrier Proteins metabolism
Histones metabolism
Nuclear Proteins chemistry
Nuclear Proteins metabolism
RNA, Messenger chemistry
RNA, Messenger metabolism
mRNA Cleavage and Polyadenylation Factors chemistry
mRNA Cleavage and Polyadenylation Factors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 52
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 23286197
- Full Text :
- https://doi.org/10.1021/bi301074r