Control of RNA Pol II Speed by PNUTS-PP1 and Spt5 Dephosphorylation Facilitates Termination by a “Sitting Duck Torpedo” Mechanism

Cortázar, Michael A.; Sheridan, Ryan M.; Erickson, Benjamin; Fong, Nova; Glover-Cutter, Kira; Brannan, Kristopher W.; Bentley, David L.

doi:10.1016/j.molcel.2019.09.031

Cited by 179 publications

(240 citation statements)

References 77 publications

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“…These results supported the idea that Spt5-CTR phosphorylation by Cdk9 is an accelerator of elongation 21,22 , whereas reversal of that phosphorylation by PP1 is a brake. Two recent reports extended this model to human cells, by showing that PP1 promotes Pol II slowing and termination through Spt5 dephosphorylation 30,31 . Another implicated PP4 in regulation of Spt5 phosphorylation and function during early stages of transcription in Caenorhabditis elegans 51 .…”

Section: Discussionmentioning

confidence: 98%

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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

show abstract

Section: Discussionmentioning

confidence: 98%

“…The effect of Cdk9 inhibition was recapitulated by an spt5 mutation that prevented Spt5-CTD phosphorylation 29 . Recently, human PP1 and its regulatory subunit PNUTS were implicated in Spt5-CTR dephosphorylation and Pol II deceleration downstream of the CPS 30,31 , suggesting conservation of this mechanism.…”

mentioning

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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“…PNUTS was initially described as a binding platform for PP1, and although several other binding partners of PNUTS have been described over time [40], the main body of knowledge on PNUTS function continues to be related to this phosphatase. The PNUTS-PP1 axis has been suggested to be responsible of DNA damage response [19], MYC activation [27], cell cycle entry [21], apoptosis [28], gene expression regulation [26] and transcription [29]. None of these studies report, however, a role for PNUTS-PP1 in physiological processes, with the exception of the work of Ciurciu and colleagues, which modelled their findings in the embryonic development of D. melanogaster.…”

Section: Discussionmentioning

confidence: 99%

“…The PNUTS-PP1 complex is formed by interaction through the canonical RxVF motif. By binding to PP1, PNUTS is implicated in the dephosphorylation and activity of several PP1 targets, such as RNA polymerase II [26], Myc [27], Rb [28] and Spt5 [29]. However, whether PNUTS plays a role in endothelial aging is not known.…”

Section: Introductionmentioning

confidence: 99%

The PNUTS-PP1 axis regulates endothelial aging and barrier function via SEMA3B suppression

Lozano-Vidal

Stanicek

Bink

et al. 2020

Preprint

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Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice presented severe multiorgan failure and vascular leakage. We showed that the PNUTS binding motif for protein phosphatase 1 (PP1) is essential to maintain endothelial barrier function. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a PP1-SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

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“…This complex, which contains PP1C, PNUTS, WDR82, and the DNA-binding protein TOX4, has a role in controlling chromatin structure (22,24). Importantly, the PTW/PP1 complex was recently been found to be a negative regulator of RNAP II elongation rate and that dephosphorylation of the transcription elongation factor Spt5 is necessary for transcription termination at polyadenylation sites in mammalian cells (25). Thus, our results indicate that GT associates with a PTW/PP1-like complex in Leishmania (which we will refer to as PJW/PP1), wherein JBP3 replaces the DNA-binding function of TOX4.…”

Section: Identification Of a Protein Complex Containing A Novel J-binmentioning

confidence: 99%

Chromatin-associated protein complexes link DNA base J and transcription termination inLeishmania

Jensen¹,

Phan²,

McDonald³

et al. 2020

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Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on post-transcriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-D-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation site of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP-tagging) and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatics analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein, which we dubbed JBP3. Down-regulation of JBP3 expression levels in Leishmania results in a substantial increase in transcriptional read-through at the 3’ end of most PTUs. Additional TAP-tagging experiments showed that JBP3 also associates with two other protein complexes. One consists of subunits with domains suggestive of a role in chromatin modification/remodeling, while the other contains subunits with similarity to those found in the PAF1C complex involved in regulation of transcription in other eukaryotes. Thus, while trypanosomatids utilize protein complexes similar to that used to control transcription termination in other eukaryotes, JBP3 appears to function as a hub linking these modules to base J, thereby enabling the parasites’ unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression.

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Control of RNA Pol II Speed by PNUTS-PP1 and Spt5 Dephosphorylation Facilitates Termination by a “Sitting Duck Torpedo” Mechanism

Cited by 179 publications

References 77 publications

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

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

The PNUTS-PP1 axis regulates endothelial aging and barrier function via SEMA3B suppression

Chromatin-associated protein complexes link DNA base J and transcription termination inLeishmania

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