A Cdk9–PP1 switch regulates the elongation–termination transition of RNA polymerase II

Parua, Pabitra K.; Booth, Gregory T.; Sansó, Miriam; Benjamin, Bradley; Tanny, Jason C.; Lis, John T.; Fisher, Robert P

doi:10.1038/s41586-018-0214-z

Cited by 132 publications

(171 citation statements)

References 43 publications

(79 reference statements)

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“…Similar to the mammalian complex, we propose PNUTS is a scaffolding protein for the entire PJW/PP1 complex and regulates PP1 function via the PP1 binding RVxF motif. Only three substrates have been identified for PNUTS/PP1: the Pol II elongation factor Spt5, the CTD of Pol II and MYC [2,60,97,98]. MYC dephosphorylation by PP1 regulates chromatin binding and stability.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids. Author summaryTrypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now PLOS Genetics | https://doi.identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including organisms that utilize polycistronic transcription and novel epigenetic marks...

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Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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“…AS alleles have frequently been used to inhibit these transcription-related kinases in budding and fission yeast (8)(9)(10)(11)(12)(13)(14)(15)(16)(17) as well as mammalian cells (18)(19)(20). Chemical inhibitors are available that target the native mammalian kinases with varying levels of specificity (21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

Selective kinase inhibition shows that Bur1 (Cdk9) phosphorylates the Rpb1 linker in vivo

Chun

Suh

Joo

et al. 2018

Preprint

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Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at Serines 5 and 7, which are known target sites, but also at Serine 2. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for Serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tSH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for this modification in vivo.

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“…Pausing and Ser2 phosphorylation (pSer2) in turn promote recruitment of factors needed for mRNA 3’-end maturation and termination 26 . We recently uncovered a regulatory circuit in fission yeast comprising Cdk9, the protein phosphatase 1 (PP1) isoform Dis2, and their common enzymatic target, Spt5, with the potential to switch Pol II from rapid elongation to a paused state permissive for termination 27 . During processive elongation, Cdk9 phosphorylates the Spt5 CTD and keeps Dis2 inactive by phosphorylating its carboxy-terminal region.…”

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“…As elongation complexes traverse the CPS, the Spt5 CTD is dephosphorylated dependent on activity of Dis2, which is a subunit of the cleavage and polyadenylation factor (CPF) 28 ; the drop in phospho-Spt5 precedes an increase in pSer2 over the 3’ pause site. Inactivation of Cdk9 or Dis2 leads to opposite effects downstream of the CPS—more rapid termination, or more extensive read-through transcription indicating a termination defect, respectively 21,27 . The effect of Cdk9 inhibition was recapitulated by an spt5 mutation that prevented Spt5-CTD phosphorylation 29 .…”

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confidence: 99%

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

Parua

Kalan

Benjamin

et al. 2020

Preprint

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Reversible phosphorylation of Pol II and accessory factors helps order the 12 transcription cycle. Here we define two kinase-phosphatase switches that operate 13 at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes 14 inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' 15 ends of genes, respectively, and have overlapping but distinct specificities for 16Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-17 proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 18 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between KOW motifs 4 19 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation 20 decreases as transcription complexes pass the cleavage and polyadenylation 21 signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in 22In fission yeast, chemical-genetic inhibition of Cdk9 led to rapid, nearly complete 130 dephosphorylation of the Spt5 CTD (T1/2 ~20 sec); the rate of decay decreased ~4-fold in 131 dis2 mutant strains, suggesting that the fast kinetics in dis2 + cells were partly due to the 132 concomitant activation of Dis2 (PP1) when Cdk9 is inactivated 27 . In HCT116 cells, both 133 pThr806 and a phosphorylation outside the CTRs, pSer666, were lost rapidly upon 134 treatment with 250 nM NVP-2 (T1/2 ~10 min), consistent with a similar, reinforcing effect 135 of kinase inhibition and phosphatase activation (Fig. 1d). 136 137

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A Cdk9–PP1 switch regulates the elongation–termination transition of RNA polymerase II

Cited by 132 publications

References 43 publications

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Selective kinase inhibition shows that Bur1 (Cdk9) phosphorylates the Rpb1 linker in vivo

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II

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