Christoph H. Borchers, Roopa Thapar, Evgeniy V. Petrotchenko, Matthew Michael Easterling, Zbigniew Dominski, and William F. Marzluff Combined top-down and bottom-up proteomics identifies a phosphorylation site in stemloop-binding proteins that contributes to high-affinity RNA binding Presented by Aliya Sadeque stemloop-binding protein Binds metazoan replication-dependent histone mRNA Not acetylated, stem-loop instead Involved in cell-cycle regulation of histone mRNA Erkmann, J. A., Wagner, E. J., Dong, J., Zhang, Y., Kutay, U. & Marzluff, W. F. (2005) Mol. Biol. Cell 16, 29602971. Previously known dSLBP is stoichiometrically phosphorylated on multiple sites Phosphatase treatment resulted in change in mobility on gel electrophoresis Previously known dSLBP is stoichiometrically phosphorylated on multiple sites Multiple phosphorylations sites at the C- terminus Based on M r determinations of intact and truncated proteins Previously known dSLBP is stoichiometrically phosphorylated on multiple sites Multiple phosphorylations sites at the C- terminus 4 phosphorylated Ser residues identified in C terminus Using Phosphatase-directed Phosphorylation-Site Determination Outline of experiments Where phosphorylation is occurring To what extent What are the biological implications? How does this affect the overall function of SLBP How does this affect RNA binding specifically? Top or Bottom? Top-down = intact protein molecular mass measurement Bottom-up = proteolytic fragment identification Strader, M. B., VerBerkmoes, N. C., Tabb, D. L., Connelly, H. M., Barton, J. W., Bruce, B. D., Pelletier, D. A., Davison, B. H., Hettich, R. L., Larimer, F. W., et al. (2004) J. Proteome Res. 3, 965978. Torres, M. P., Marzluff, W. F.&Borchers, C. H. (2005) J. Proteome Res. 4, 1628 1635. Top-down FTICR analysis of M r of RBD and RPD Single major molecular species for RBD 9 Da lower than predicted | | | | Top-down FTICR analysis of M r of RBD and RPD Two ion series for RPD Top-down Collision-induced fragmentation of 4_P ions followed by M r analysis of fragments Sequence tag carfuffle N-term sequence tags did not correspond to predicted b-ions..?? Top-down De novo sequencing by CID revealed Modified N-terminus Top-down De novo sequencing by CID revealed Modified N-terminus Removal of N-term Met Top-down De novo sequencing by CID revealed Modified N-terminus Removal of N-term Met Additional P on T230 Top-down De novo sequencing by CID revealed Modified N-terminus Removal of N-term Met Additional P on T230 9 Da discrepancy loss of N-term Met (-131 Da) Acetylation of new N-term Ser (+42 Da) Additional Phosphorylation (+80 Da) = 9Da Confirmed by analysis of tryptic digests of dSLBP Top-down Stoichiometric phosphorylation of T230 Baculovirus expressed dSLBP Bottom-up Stoichiometric phosphorylation of T230 Baculovirus expressed dSLBP dSLBP-RPD from baculovirus and full- length dSLBP from Drosophila Top-down Stoichiometric phosphorylation of T230 M r analysis of corresponding hSLBP T171 Same 9 Da discrepancy Same tag carfuffle So now we know where they are Big ups Effects on RNA Binding Effect on RNA Binding Treated with calf intestinal phosphatase Full-length hSLBP hSLBP-RBD Used T230A mutant Full-length dSLBP dSLBP-RPD Effect on RNA Binding Gel mobility shift assay Shows that they are binding RNA Effect on RNA Binding Filter-binding assay to measure affinity Dephosphorylation results in up to 10-fold lower affinity T230 Non-viable null mutant dSLBP is not rescued by T230A Mutations that affect mRNA-SLBP affinity result in low expression of mRNA Sea urchin TPNK studies: unable to phosphorylate Thr Binding Mechanism One or more dSLBP phosphoryl groups contributes favorably to overall stability of the SLBP-RNA complex. Sketchiness (a.k.a. critique) Why use top down at all Insect cells - are they sure PTMs were done properly? Filter binding assays assume 1:1 stoichiometry.why? Why even use this method? Fraction bound curves: fit to standard is a stretch although mutant still worse Acetylationisnt that a big deal? How do these results reflect structural stability? Extent of phosphorylation - cell cycle regd?