Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Czudnochowski, Nadine; Bösken, Christian A.; Geyer, Matthias

doi:10.1038/ncomms1846

Cited by 167 publications

(191 citation statements)

References 60 publications

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“…2, Supplementary Table 2), with incomplete phosphorylation of the C-terminal repeat, and two internal repeats lacking a Ser5-Pro6 pair (Ser5 in YSPSSSN and Thr5 in YTPVTPS). The observed rates are consistent with a distributive mechanism similar to that observed for the human P-TEFb 12 . Thus the in vitro phosphorylation reactions produce a nearly complete hyper-pSer5 state, in agreement with our MS data (Fig.…”

Section: Transgenic Flies Reveal a Ctd Region Needed For Developmentsupporting

confidence: 87%

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Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

Showalter

Gibbs

2017

Biophysical Journal

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The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. Here we identify a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalize on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms.

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Section: Transgenic Flies Reveal a Ctd Region Needed For Developmentsupporting

confidence: 87%

“…2a,c). Preferential phosphorylation in vitro of Ser5 over other amino acids in the CTD by human P-TEFb has been previously observed 12,13 .…”

Section: Transgenic Flies Reveal a Ctd Region Needed For Developmentmentioning

confidence: 77%

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

Showalter

Gibbs

2017

Biophysical Journal

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“…In addition, eukaryotic gene expression is regulated by RNAPII through the phosphorylation of its carboxyl-terminal domain (CTD) (35). Ser-2 phosphorylation (2P) marks the elongation state, whereas Ser-5 is phosphorylated (5P) in the initiation phase (36). Therefore, we examined whether Menin is required for the recruitment of RNAPII to the Il17a gene locus in Th17 cells.…”

Section: Menin Does Not Control the Expression Of Other Key Transcripmentioning

confidence: 99%

Trithorax complex component Menin controls differentiation and maintenance of T helper 17 cells

Watanabe¹,

Onodera²,

Kanai³

et al. 2014

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

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Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin −/− T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression.

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“…We focused on Hexim1 F208Bpa, because this position has the highest cross-linking efficiency in human cell extracts and it belongs to an evolutionary-conserved peptide sequence ( 202 PYNTTQFLM 210 ), the PYNT motif, which was previously shown to be involved in P-TEFb inhibition (20,21,26). P-TEFb was added to purified F208Bpa Hexim1.Larp7.7SK RNA complex to reconstitute an inactive P-TEFb.Hexim1.Larp7.7SK RNA complex.…”

Section: Resultsmentioning

confidence: 99%

“…1A, green). Furthermore, an evolutionary conserved motif in the center of the protein, the "PYNT" sequence, is critical for P-TEFb binding and inhibition (20,21,26). This motif, distinct from the cyclin T-binding domain, is located between a basic region (BR) involved in 7SK RNA binding and an acidic region (AR) (Fig.…”

mentioning

confidence: 99%

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

Kobbi

Demey-Thomas

Brayé

et al. 2016

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

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The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide ( 202 PYNTTQFLM 210 ) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate bindingsite. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.cyclin-dependent kinase inhibition | transcription factor regulation | regulatory noncoding RNA | benzoyl phenylalanine | protein-protein cross-linking

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Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Cited by 167 publications

References 60 publications

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

Trithorax complex component Menin controls differentiation and maintenance of T helper 17 cells

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

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