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 stem–loop-binding proteins that contributes to high-affinity RNA binding Presented by Aliya Sadeque

stem–loop-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, 2960–2971.

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, 965–978.

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  Acetylation…isn’t that a big deal?  How do these results reflect structural stability?  Extent of phosphorylation - cell cycle reg’d?