Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem–loop-binding proteins that contributes to high-affinity RNA binding

Borchers, Christoph H.; Thapar, Roopa; Petrotchenko, Evgeniy V.; Torres, Matthew P.; Speir, J. Paul; Easterling, Michael L.; Domiński, Zbigniew; Marzluff, William F.

doi:10.1073/pnas.0511289103

Cited by 43 publications

(69 citation statements)

References 38 publications

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“…S1A). dSLBP-EPNK bound to stem-loop RNA threefold more tightly than nonphosphorylated dSLBP RPD-WT, in accordance with a previous report where phosphorylation of the TPNK motif increased binding affinity sevenfold (16). dSLBP RPD-4E bound to stemloop RNA 12-fold more tightly than did dSLBP RPD-WT.…”

Section: Resultssupporting

confidence: 91%

“…This RNAprocessing domain (RPD) is necessary and sufficient for histone mRNA 3′-end processing in vitro (15). The RBDs of human SLBP (hSLBP) and Drosophila SLBP (dSLBP) are phosphorylated at a Thr residue in a conserved TPNK motif (16,17). The recent crystal structure of hSLBP RBD in complex with histone mRNA stem loop and 3′ hExo, a 3′-5′ exonuclease required for histone mRNA degradation, provided the first molecular insights into the architecture of this complex, and revealed how the hSLBP RBD forms a new RNA-binding motif to interact with the stem-loop RNA (18).…”

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confidence: 99%

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Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

Zhang

Tan

DeRose

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.X-ray crystallography | NMR | intrinsically disordered protein H istone synthesis increases at the beginning of S-phase to package newly replicated DNA with histone proteins, but synthesis must be shut down rapidly and histone mRNA degraded at the end of DNA replication because of the toxicity of surplus histone proteins (1, 2). This cyclic demand for histones requires strict regulation, which is achieved mainly by controlling the synthesis and degradation of histone mRNA (3). Replication-dependent histone mRNAs are the only known cellular mRNAs that are not polyadenylated and instead end with a conserved stem loop (4). Histone mRNAs are generated from longer histone pre-mRNAs as a result of an endonucleolytic cleavage between the stem loop and a purine-rich downstream sequence termed the "histone downstream element" (HDE) (5).Stem-loop-binding protein (SLBP), also known as "hairpinbinding protein" (6), binds to the histone mRNA stem loop, and U7 small nuclear ribonucleoprotein binds to the HDE (7). Other factors, including the endonuclease CPSF-73, are involved in both polyadenylation and histone mRNA 3′-end processing (8-11). In mammalian nuclear extracts, SLBP is not absolutely required for the biochemical reaction of processing (12). In contrast, cleavage of histone pre-mRNA in Drosophila cells and nuclear extracts requires the binding of SLBP to the stem loop (10, 13).The minimal histone mRNA processing domain of Drosophila SLBP contains a 72-aa RNA-binding domain (RBD) unique to SLBPs and an 18-aa C-terminal region (Fig. 1A) (14). This RNAprocessing domain (RPD) is necessary and sufficient for histone mRNA 3′-end processing in vitro (15). The RBDs of human SLBP (hSLBP) and Drosophila SLBP (dSLBP) are phosphorylated at a Thr residue in a conserved TPNK motif (16,17). The recent crystal structure of hSLBP RBD in complex with histone mRNA stem loop and 3′ hExo, a 3′-5′ exonuclease required for histone mRNA degradation, provided the first molecular insights into the architecture of this complex, and revealed how the hSLBP RBD forms a new ...

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Section: Resultssupporting

confidence: 91%

mentioning

confidence: 99%

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

Zhang

Tan

DeRose

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, the fluorescence anisotropy and the mobility shift assays suggest that Pin1 and a phosphatase such as PP2A can act to increase the off-rate of SLBP from the stem-loop at the 3= end of histone mRNA, allowing exchange of labeled and unlabeled RNA in the complex. The results are consistent with our published studies (3,65) showing that SLBP that is dephosphorylated at Thr171 has a higher off-rate for the histone mRNA stem-loop.…”

Section: Structural Basis For the Phosphorylated Slbp-pin1 Interactionsupporting

confidence: 93%

“…We have recently shown that SLBP undergoes proline isomerization about a conserved phosphorylated Thr-Pro sequence in its RNA binding domain (RBD) (3,29,65). There are six Ser-Pro/Thr-Pro sequences in human SLBP, all of which have been previously shown to be phosphorylated by mass spectrometry (12).…”

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confidence: 99%

The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

Krishnan

Lam

Fritz

et al. 2012

Molecular and Cellular Biology

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f Histone mRNAs are rapidly degraded at the end of S phase, and a 26-nucleotide stem-loop in the 3= untranslated region is a key determinant of histone mRNA stability. This sequence is the binding site for stem-loop binding protein (SLBP), which helps to recruit components of the RNA degradation machinery to the histone mRNA 3= end. SLBP is the only protein whose expression is cell cycle regulated during S phase and whose degradation is temporally correlated with histone mRNA degradation. Here we report that chemical inhibition of the prolyl isomerase Pin1 or downregulation of Pin1 by small interfering RNA (siRNA) increases the mRNA stability of all five core histone mRNAs and the stability of SLBP. Pin1 regulates SLBP polyubiquitination via the Ser20/Ser23 phosphodegron in the N terminus. siRNA knockdown of Pin1 results in accumulation of SLBP in the nucleus. We show that Pin1 can act along with protein phosphatase 2A (PP2A) in vitro to dephosphorylate a phosphothreonine in a conserved TPNK sequence in the SLBP RNA binding domain, thereby dissociating SLBP from the histone mRNA hairpin. Our data suggest that Pin1 and PP2A act to coordinate the degradation of SLBP by the ubiquitin proteasome system and the exosomemediated degradation of the histone mRNA by regulating complex dissociation.

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“…A common design goal in recent instruments is the improvement of both duty cycle and dynamic range through selective accumulation of ions from the source during the time necessary to record the FT-ICR spectrum. Q/FT-ICR instruments are now available commercially from two vendors, Bruker Daltonics [67] and Varian [68].…”

Section: Beam-trap Hybrid Instrumentsmentioning

confidence: 99%

Hybrid mass spectrometers for tandem mass spectrometry

Glish

Burinsky

2008

J. Am. Soc. Mass Spectrom.

101

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Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis in tandem mass spectrometry (MS/MS) experiments are often referred to as "hybrid" mass spectrometers. The general goal in the design of a hybrid instrument is to combine different performance characteristics offered by various types of analyzers into one mass spectrometer. These performance characteristics may include mass resolving power, the ion kinetic energy for collision-induced dissociation, and speed of analysis. This paper provides a review of the development of hybrid instruments over the last 30 years for analytical applications. (J Am Soc Mass Spectrom 2008, 19, 161-172) © 2008 American Society for Mass Spectrometry T andem mass spectrometry (MS/MS), in a very generic description, is a process in which an ion formed in an ion source is mass-selected in the first stage of analysis, reacted, and then the charged products from the reaction are analyzed in the second stage of analysis. The type and quality of data that is obtained can vary greatly depending upon the type of analyzer used in the first and second stages of analysis, and the type of reaction performed between the stages of analysis. The reactions that can be done also can depend upon the type of analyzer. Over the years there have been a variety of means developed to measure the mass-to-charge ratio of gas-phase ions. The most common methods involve: dispersion based on ion momentum or kinetic energy (magnetic and electric sector instruments); separation in time based on ion velocity (time-of-flight); transmission through an electrodynamic field (quadrupole mass filter); and periodic motion in a magnetic or electrodynamic field (ion traps). There are differences in the experimental parameters associated with these various analysis methods that are pertinent to the MS/MS experiment. Some parameters are obvious, typically related to the performance of the mass analyzer while others are more subtle, related to the reactions/chemistry occurring between the stages of analysis. Many times these different parameters are used to categorize MS/MS experiments.One parameter is the ion kinetic energy. Sector and time-of-flight (TOF) instruments typically operate at "high" ion kinetic energies (5-20 keV), whereas "low" ion kinetic energies (Ͻ50 eV) are typical in quadrupole mass filters and ion traps. The ion kinetic energy is an important parameter in MS/MS experiments because the most common reaction involves colliding the ion of interest with a target gas atom or molecule. When performing ion-neutral collision experiments, the possible reactions that can be accessed (e.g., collisioninduced dissociation, collisional cooling, charge permutation, ion/molecule reaction) depends upon the ion kinetic energy. The appearance of the MS/MS spectrum can change drastically as a function of the collision (ion kinetic) energy. A related factor that is equally important, but often not considered, is the time frame of the experiment, that is, the elapsed time bet...

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Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem–loop-binding proteins that contributes to high-affinity RNA binding

Cited by 43 publications

References 38 publications

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

Hybrid mass spectrometers for tandem mass spectrometry

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