Mapping the Phosphorylation Pattern of <i>Drosophila melanogaster</i> RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry

Mayfield, Joshua E.; Robinson, Michelle; Cotham, Victoria C.; Irani, Seema; Matthews, Wendy L.; Ram, Anjana; Gilmour, David S.; Cannon, Joe R.; Zhang, Yan Jessie; Brodbelt, Jennifer S.

doi:10.1021/acschembio.6b00729

Cited by 26 publications

(62 citation statements)

References 47 publications

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“…10–12 Higher-energy collisional activation (HCD) has somewhat addressed this challenge, 13 but alternative fragmentation methods, including electron transfer dissociation (ETD) and several photo-dissociation approaches, have also proven valuable for phosphoproteomic experiments. 14–24 …”

mentioning

confidence: 99%

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Riley¹,

Hebert²,

Dürnberger

et al. 2017

Anal. Chem.

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The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90-minute analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photo-activation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, a ~23.5 kilodalton phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.

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mentioning

confidence: 99%

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Riley¹,

Hebert²,

Dürnberger

et al. 2017

Anal. Chem.

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“…2E, gold trace). To identify the position of phosphorylation with single-residue resolution, we applied UV photodissociation (UVPD) using 193-nm photons as an alternative to existing collision-and electron-based activation methods for tandem MS analysis (30). UVPD provides several compelling advantages in the context of MS/MS analysis of the CTD (31).…”

Section: Tyr 1 Determines the Specificity Of Ctd Phosphatase Ssu72mentioning

confidence: 99%

“…GST-CTD proteins were expressed and purified as described in established protocols published previously (30). Concisely, the cell pellet was lysed by sonication in a buffer at pH 8.0 containing 500 mM NaCl, 0.1% Triton X-100, and 10% glycerol.…”

Section: Tyr 1 Determines the Specificity Of Ctd Phosphatase Ssu72mentioning

confidence: 99%

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription

Irani

Sipe

Yang

et al. 2019

Journal of Biological Chemistry

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Edited by John M. Denu The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser 5 (pSer 5) is gradually dephosphorylated by CTD phosphatases, whereas Ser 2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer 5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer 2 and pSer 5 CTDs. We found that Ssu72 dephosphorylates pSer 5 effectively but only has low activities toward pSer 7 and pSer 2. The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ␤-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr 1 next to Ser 2 , prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr 1 further prohibited Ssu72 binding to pSer 2 and thereby prevented untimely Ser 2 dephosphorylation. Our results reveal critical roles for Tyr 1 in differentiating the phosphorylation states of Ser 2 / Ser 5 of CTD in RNA polymerase II that occur at different stages of transcription.

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“…Theoretically, the sets of the combinations for just the Ser2 and Ser5 two sites on the large number of the CTD repeats could be extremely big. 34 The recently developed genetic-or ultraviolet photodissociation-based mass-spectrometric approaches [35][36][37] have the potential of revealing the exact CTD phosphorylation patterns that are modulated by GTPase signaling in response to distinct extracellular signals.…”

Section: Important Questions To Be Addressed In the Futurementioning

confidence: 99%

Ras and Rho GTPase regulation of Pol II transcription: A shortcut model revisited

Zheng

2017

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Transcriptional control is critical in relaying signals mediated by Ras and Rho family small GTPases to effect gene expression. In the classical model, signaling components such as MAP kinase target sequence-specific transcription factors, which in turn recruit RNA polymerase (Pol) II holoenzyme to the promoter and activate transcription. Findings in recent years have led to the proposal of a shortcut model in which the Mediator components of the Pol II holoenzyme are regulated by signaling pathways. A very recent finding shows that an evolutionarily conserved Rho GTPase signaling pathway can directly modulate the Pol II C-terminal domain (CTD) phosphorylation by inhibiting the CTD phosphatase in yeast and Arabidopsis. This shortcut model allows direct targeting of the Pol II CTD code and thus has an advantage over the classical model in bringing about rapid, large-scale changes in gene expression.

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Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry

Cited by 26 publications

References 47 publications

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription

Ras and Rho GTPase regulation of Pol II transcription: A shortcut model revisited

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