Rtr1 Is a Dual Specificity Phosphatase That Dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD

doi:10.1016/j.jmb.2014.06.010

Journal of Molecular Biology

Volume 426, Issue 16, 12 August 2014, Pages 2970-2981

https://doi.org/10.1016/j.jmb.2014.06.010 Get rights and content

Highlights

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Rtr1 is a new phosphatase unrelated structurally to known such enzymes.
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Rtr1's N-terminal domain represents a novel fold for phosphatase function.
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Disruption of in vitro activity displays gene deletion phenotypes in vivo.
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The C-terminal region of Rtr1 inhibits phosphatase activity.
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Rtr1 selectively dephosphorylates both Tyr1 and Ser5 on the PolII CTD.

Abstract

The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only serine 5 on the CTD but also the newly described anti-termination tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.

Graphical abstract

Introduction

The phosphorylation state of the C-terminal domain (CTD) of RNA polymerase II (PolII) Rpb1 subunit controls transcription [1]. The CTD consists of a highly conserved heptapeptide (Y₁S₂P₃T₄S₅P₆S₇) repeated between 26 times in Saccharomyces cerevisiae and 52 times in humans. Ser2 and Ser5 are reversibly phosphorylated, while the prolines are subject to cis–trans isomerization facilitated by isomerases such as Ess1 [2], [3], [4], [5], [6]. In addition, the Tyr1, Thr4 and Ser7 residues can also be phosphorylated, although the impact and scope of these modifications is less well understood [7], [8], [9]. The dynamic combination of post-translational modifications constitutes a “CTD code” that helps recruit or activate various factors to the polymerase during the transcription cycle [10], [11], [12].

High levels of phosphorylation of Ser5 (Ser5P) on the CTD occur at or near the promoter and help recruit mRNA capping and transcription elongation factors [13], [14], [15]. This modification can also act as a signal for the snoRNA/snRNA termination pathway via the Nrd1-Nab3-Sen1 complex in yeasts [16]. Ser5P is progressively dephosphorylated as the polymerase progresses into the elongation and termination phases of transcription. In contrast, Ser2 phosphorylation (Ser2P) levels are low at the start of transcription and increase as the polymerase moves along a gene, where this modification signals the recruitment and/or activation of transcription termination factors [17], [18], [19].

Multiple Ser2/5 kinases and phosphatases have been identified [1], but the identity of the phosphatase responsible for the critical transition from Ser5P to Ser2P during transcriptional elongation remains unclear. Yeast Rtr1, a highly conserved protein in all eukaryotes (Fig. S1), was recently proposed to be the Ser5P phosphatase responsible for this transition [20], a hypothesis further supported by the independent observation that its human ortholog (RPAP2) has phosphatase activity with identical selectivity profile: active on Ser5P, but not upon Ser2P nor Ser7P [20], [21]. However, this attribution was negated by the lack of in vitro phosphatase activity in Kluyveromyces lactis Rtr1, whose crystal structure also failed to reveal a canonical active site observed in other phosphatases [22]. It was proposed that the phosphatase activity detected for Rtr1 might arise from the co-purification of an Escherichia coli phosphatase enzyme, although it would appear unlikely that the accidental presence of a recombinant protein from bacterial sources would yield an enzyme that selectively dephosphorylates a substrate without an equivalent in bacteria.

Here we resolve this controversy by reporting that Rtr1 is active as a phosphatase and that its enzymatic activity is functional: mutation in a single absolutely conserved residue that significantly reduces catalytic activity in vitro also abolishes its function in vivo. We further show that Rtr1 can target and dephosphorylate PolII CTD repeats carrying both Ser5P and the newly described anti-termination Tyr1 phosphorylation marker, providing additional evidence that Rtr1 is the phosphatase that promotes the transition from initiation to the elongation and termination phases of transcription.

Section snippets

Rtr1 is a phosphatase

We independently determined the crystal structure of the K. lactis Rtr1 (KlRtr1) N-terminal domain (NTD) (amino acids 1–156; Table 1), which is nearly identical with the previously determined structure [22] (C^α RMSD = 0.35 Å) (Fig. 1a). Purification of the full-length KlRtr1 protein using standard protocols (Fig. 1b, upper flow) resulted in preparations that lacked activity when assayed against both phosphorylated GST-CTD (GST, glutathione S-transferase) (data not shown) and the acid phosphatase

Discussion

The interplay of kinases and phosphatases that act upon the CTD of RNA PolII regulates and times the synthesis and biogenesis of cellular RNAs. However, the identity of the critical transition phosphatase that removes the Ser5P marker and shift the polymerase to the elongation and termination mode remains to be firmly established. Rtr1 (RPAP2 in vertebrates), a highly conserved protein in all eukaryotes, was proposed to be such a phosphatase in two independent studies showing that Rtr1 in both

Protein expression and purification

S. cerevisiae (Sc) and K. lactis (Kl) Rtr1 proteins, as well as human RPAP2 (1–189), were cloned into a modified pET-28a (Novagen) vector with a Protein G B1 domain (GB1) fused to the N-terminus to facilitate expression. Plasmids encoding the gene were transformed into Rosetta DE3 E. coli, shaken at 37 °C until induction with IPTG and expressed overnight at 18 °C. Cells were harvested the next morning and resuspended in lysis buffer [50 mM Hepes (pH 7.5), 200 mM NaCl, 30 mM imidazole and 5 mM βME],

Acknowledgements

The authors would like to thank the staff at the Advanced Light Source (Berkeley, CA) for assistance in data collection. We would also like to thank the Stoddard laboratory at the Fred Hutchinson Cancer Research Center for providing access to their home X-ray source for initial crystal screening. We thank Dr. Ning Zheng for his advice and guidance in solving the crystal structure of KlRtr1 NTD. Chromotek generously allowed us early access to the anti-Tyr1P antibody. TFIIH was kindly provided by

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Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.
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First, Hsu et al.[77] identified a point mutation in Rtr1 (E66A) that impairs phosphatase activity in vitro, ruling out the possibility of a contaminant being responsible for its catalytic activity. Importantly, the E66A mutant gives rise to a growth defect similar to RTR1 deletion in vivo[77], demonstrating that the phosphatase activity is relevant for Rtr1's function. Second, a recent crystal structure of the full-length Rtr1 from Saccharomyces cerevisiae allowed for the identification of the active site missing from the K. lactis structure (Yan Jessie Zhang, personal communication).
The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this “Perspective,” we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed.
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The phosphorylation state of the C-terminal domain of RNA polymerase II is required for the temporal and spatial recruitment of various factors that mediate transcription and RNA processing throughout the transcriptional cycle. Therefore, changes in CTD phosphorylation by site-specific kinases/phosphatases are critical for the accurate transmission of information during transcription. Unlike kinases, CTD phosphatases have been traditionally neglected as they are thought to act as passive negative regulators that remove all phosphate marks at the conclusion of transcription. This over-simplified view has been disputed in recent years and new data assert the active and regulatory role phosphatases play in transcription. We now know that CTD phosphatases ensure the proper transition between different stages of transcription, balance the distribution of phosphorylation for accurate termination and re-initiation, and prevent inappropriate expression of certain genes. In this review, we focus on the specific roles of CTD phosphatases in regulating transcription. In particular, we emphasize how specificity and timing of dephosphorylation are achieved for these phosphatases and consider the various regulatory factors that affect these dynamics.
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