An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape

Das, Apurba K.; Banday, Mahrukh; Fisher, Michael A.; Chang, Yen‐Hsiang; Rosenfeld, Jeffrey; Bellofatto, Vivian

doi:10.1093/nar/gkx486

Cited by 18 publications

(14 citation statements)

References 34 publications

(51 reference statements)

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“…The CTD of Pol II in trypanosomatids is unique in that it does not contain the heptad or other repetitive motifs that are conserved from yeast to humans [101]. However, the CTD was shown to be essential for Pol II transcription in T. brucei [102,103] and 17 phosphorylated sites have been identified within the CTD [100]. Studies also suggest that CTD phosphorylation is required for Pol II association with trypanosome chromatin [104].…”

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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“…The CTD of Pol II in trypanosomatids is unique in that it does not contain the heptad or other repetitive motifs that are conserved from yeast to humans (95). However, the CTD was shown to be essential for Pol II transcription in T. brucei (96, 97) and 17 phosphorylated sites have been identified within the CTD (94). Studies also suggest that CTD phosphorylation is required for Pol II association with trypanosome chromatin (98).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Sabatini

Kieft

Zhang

et al. 2019

Preprint

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19 Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA 20 Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how 21 specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription 22 termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-23 glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass 24 spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein 25 (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate 26 JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, 27 JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian 28 PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. 29 Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of 30 PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in 31 Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, 32 suggesting an additional role of the complex in regulating termination of bi-directional transcription. In 33 addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for 34 host immune evasion. Our results provide the first direct mechanistic link between base J and regulation 35 of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating 36 polycistronic transcription in trypanosomatids. 37 38 Author Summary 39 Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping 40 sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain 41 multiple genes that are co-transcribed from a single promoter. We have recently described the presence 42 of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene 43 clusters where the loss of base J and H3.V leads to read-through transcription and the expression of 44 downstream genes. We now identify a novel stable multimeric complex containing a J binding protein . CC-BY 4.0 International license is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint 45 (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) 46 and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved 47 in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead 48 to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex reg...

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“…The CTD of trypanosome RPB1 does not carry the heptad or other repetitive motifs although 17 phospho‐sites have been identified (Nett et al, 2009; Urbaniak, Martin, & Ferguson, 2013). While this non‐canonical CTD was shown to be important for RNA pol II function (Das et al, 2017; Das & Bellofatto, 2009), the functional significance of its phosphorylation is not understood.…”

Section: Introductionmentioning

confidence: 99%

Rapid block of pre‐mRNA splicing by chemical inhibition of analog‐sensitive CRK9 in Trypanosoma brucei

Gosavi

Srivastava

Badjatia

et al. 2020

Molecular Microbiology

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Trypanosoma brucei CRK9 is an essential cyclin‐dependent kinase for the parasite‐specific mode of pre‐mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans‐splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre‐mRNAs and the disappearance of the trans‐splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog‐sensitive CRK9 (CRK9AS). Inhibition of CRK9AS in these cells by the ATP‐competitive inhibitor 1‐NM‐PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre‐mRNA processing. Since defective trans‐splicing was detected as early as 5 min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre‐mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12–24 hr. Surprisingly, RNA pol II‐mediated RNA synthesis in 24 hr‐treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.

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An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape

Cited by 18 publications

References 34 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

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Rapid block of pre‐mRNA splicing by chemical inhibition of analog‐sensitive CRK9 in Trypanosoma brucei

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