Electrostatic contribution of serine phosphorylation to the Drosophila SLBP--histone mRNA complex.
Unlike all other metazoan mRNAs, mRNAs encoding the replication-dependent histones are not polyadenylated but end in a unique 26 nucleotide stem-loop structure. The protein that binds the 3' end of histone mRNA, the stem-loop binding protein (SLBP), is essential for histone pre-mRNA processing, mRNA translation, and mRNA degradation. Using biochemical, biophysical, and nuclear magnetic resonance (NMR) experiments, we report the first structural insight into the mechanism of SLBP-RNA recognition. In the absence of RNA, phosphorylated and unphosphorylated forms of the RNA binding and processing domain (RPD) of Drosophila SLBP (dSLBP) possess helical secondary structure but no well-defined tertiary fold. Drosophila SLBP is phosphorylated at four out of five potential serine or threonine sites in the sequence DTAKDSNSDSDSD at the extreme C-terminus, and phosphorylation at these sites is necessary for histone pre-mRNA processing. Here, we provide NMR evidence for serine phosphorylation of the C-terminus using (31)P direct-detect experiments and show that both serine phosphorylation and RNA binding are necessary for proper folding of the RPD. The electrostatic effect of protein phosphorylation can be partially mimicked by a mutant form of SLBP wherein four C-terminal serines are replaced with glutamic acids. Hence, both RNA binding and protein phosphorylation are necessary for stabilization of the SLBP RPD.
Thapar
Roopa
R
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Marzluff
William F
WF
Redinbo
Matthew R
MR
D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D006657:Histones / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity
D029721:Drosophila Proteins; D006657:Histones; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C490155:SLBP protein, Drosophila; D012694:Serine