Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding.
The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, 27599, USA. christoph_borchers@med.unc.edu
Christoph H
CH
Borchers
Roopa
R
Thapar
Evgeniy V
EV
Petrotchenko
Matthew P
MP
Torres
J Paul
JP
Speir
Michael
M
Easterling
Zbigniew
Z
Dominski
William F
WF
Marzluff
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C490155:SLBP protein, Drosophila; C092663:SLBP protein, human; D039221:mRNA Cleavage and Polyadenylation Factors; D010769:Phosphothreonine; D012313:RNA