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

2019 ◽  
Author(s):  
Rudo Kieft ◽  
Yang Zhang ◽  
Alexandre P. Marand ◽  
Jose Dagoberto Moran ◽  
Robert Bridger ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Protein ◽  
Transcription Termination ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Pol Ii ◽  
Polycistronic Transcription ◽  
Dna Base ◽  
Multimeric Complex

AbstractBase 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 promoters, 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 important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.Author SummaryTrypanosoma brucei is an early-diverged 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 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 divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

scholarly journals KH-type splicing regulatory protein is a new component of chromatoid body

Reproduction ◽  
2017 ◽  
Vol 154 (6) ◽  
pp. 723-733 ◽  
Author(s):  
Huijuan Zhang ◽  
Guishuan Wang ◽  
Lin Liu ◽  
Xiaolin Liang ◽  
Yu Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Germ Cell ◽  
Knockout Mice ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Somatic Cells ◽  
Chromatoid Body ◽  
Mechanistic Insight ◽  
Insight Into

The chromatoid body (CB) is a specific cloud-like structure in the cytoplasm of haploid spermatids. Recent findings indicate that CB is identified as a male germ cell-specific RNA storage and processing center, but its function has remained elusive for decades. In somatic cells, KH-type splicing regulatory protein (KSRP) is involved in regulating gene expression and maturation of select microRNAs (miRNAs). However, the function of KSRP in spermatogenesis remains unclear. In this study, we showed that KSRP partly localizes in CB, as a component of CB. KSRP interacts with proteins (mouse VASA homolog (MVH), polyadenylate-binding protein 1 (PABP1) and polyadenylate-binding protein 2 (PABP2)), mRNAs (Tnp2 and Odf1) and microRNAs (microRNA-182) in mouse CB. Moreover, KSRP may regulate the integrity of CB via DDX5-miRNA-182 pathway. In addition, we found abnormal expressions of CB component in testes of Ksrp-knockout mice and of patients with hypospermatogenesis. Thus, our results provide mechanistic insight into the role of KSRP in spermatogenesis.

scholarly journals Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation

2021 ◽  
Author(s):  
Hee-Sook Kim
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Transcription Termination ◽  
Cell Cycle Control ◽  
Histone Variants ◽  
Transcription Start ◽  
Compensatory Mechanisms ◽  
Polycistronic Transcription ◽  
Secondary Function ◽  
Transcription And Replication

In Trypanosoma brucei, genes assemble into polycistronic transcription units (PTUs). Transcription termination sites (TTSs) hold deposition sites for three non-essential chromatin factors, histone variants (H3v and H4v) and a DNA modification (base J, a hydroxyl-glucosyl dT). Here, I found that H4v is a major sign for transcription termination at TTSs and readthrough transcription machineries progress until they encounter the next bidirectional transcription start site. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J deficiency, accompanied by accumulation of sub-G1 population. Base J likely contributes to DNA replication and cell-cycle control. I propose that the coordinated actions of H3v, H4v and J function in concert for cellular fate determination and provide compensatory mechanisms for each other in chromatin organization, transcription, and replication.

scholarly journals Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity

2016 ◽  
Vol 310 (11) ◽  
pp. C1024-C1036 ◽  
Author(s):  
Jonathan M. Memme ◽  
Ashley N. Oliveira ◽  
David A. Hood
Keyword(s):  
Endoplasmic Reticulum ◽  
Contractile Activity ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Peroxisome Proliferator ◽  
Tauroursodeoxycholic Acid ◽  
Protein Levels ◽  
Activating Transcription Factor 4 ◽  
Exercise Induced

The mitochondrial and endoplasmic reticulum unfolded protein responses (UPRmt and UPRER) are important for cellular homeostasis during stimulus-induced increases in protein synthesis. Exercise triggers the synthesis of mitochondrial proteins, regulated in part by peroxisome proliferator activator receptor-γ coactivator 1α (PGC-1α). To investigate the role of the UPR in exercise-induced adaptations, we subjected rats to 3 h of chronic contractile activity (CCA) for 1, 2, 3, 5, or 7 days followed by 3 h of recovery. Mitochondrial biogenesis signaling, through PGC-1α mRNA, increased 14-fold after 1 day of CCA. This resulted in 10–32% increases in cytochrome c oxidase activity, indicative of mitochondrial content, between days 3 and 7, as well as increases in the autophagic degradation of p62 and microtubule-associated proteins 1A/1B light chain 3A (LC3)-II protein. Before these adaptations, the UPRER transcripts activating transcription factor-4, spliced X-box-binding protein 1, and binding immunoglobulin protein were elevated (1.3- to 3.8-fold) at days 1–3, while CCAAT/enhancer-binding protein homologous protein (CHOP) and chaperones binding immunoglobulin protein and heat shock protein (HSP) 70 were elevated at mRNA and protein levels (1.5- to 3.9-fold) at days 1–7 of CCA. The mitochondrial chaperones 10-kDa chaperonin, HSP60, and 75-kDa mitochondrial HSP, the protease ATP-dependent Clp protease proteolytic subunit, and the regulatory protein sirtuin-3 of the UPRmt were concurrently induced 10–80% between days 1 and 7. To test the role of the UPR in CCA-induced remodeling, we treated animals with the endoplasmic reticulum stress suppressor tauroursodeoxycholic acid and subjected them to 2 or 7 days of CCA. Tauroursodeoxycholic acid attenuated CHOP and HSP70 protein induction; however, this failed to impact mitochondrial remodeling. Our data indicate that signaling to the UPR is rapidly activated following acute contractile activity, that this is attenuated with repeated bouts, and that the UPR is involved in chronic adaptations to CCA; however, this appears to be independent of CHOP signaling.

scholarly journals Functional Characterization of a Trypanosoma brucei TATA-Binding Protein-Related Factor Points to a Universal Regulator of Transcription in Trypanosomes

2004 ◽  
Vol 24 (21) ◽  
pp. 9610-9618 ◽  
Author(s):  
Jia-peng Ruan ◽  
George K. Arhin ◽  
Elisabetta Ullu ◽  
Christian Tschudi
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Protein ◽  
Functional Characterization ◽  
Tata Binding Protein ◽  
Related Factor ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol I ◽  
Rna Genes

ABSTRACT Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3′ portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

scholarly journals Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei

2021 ◽  
Vol 9 ◽  
Author(s):  
Hee-Sook Kim
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Genetic Interaction ◽  
Transcription Termination ◽  
Genome Structure ◽  
Histone Variants ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Compensatory Mechanisms ◽  
Polycistronic Transcription

In Trypanosoma brucei, genes are assembled in polycistronic transcription units (PTUs). Boundaries of PTUs are designated transcription start sites and transcription termination sites (TTSs). Messenger RNAs are generated by trans-splicing and polyadenylation of precursor RNAs, and regulatory information in the 3′ un-translated region (UTR), rather than promoter activity/sequence-specific transcription factors, controls mRNA levels. Given this peculiar genome structure, special strategies must be utilized to control transcription in T. brucei. TTSs are deposition sites for three non-essential chromatin factors—two of non-canonical histone variants (H3v and H4v) and a DNA modification (base J, which is a hydroxyl-glucosyl dT). This association generated the hypothesis that these three chromatin marks define a transcription termination site in T. brucei. Using a panel of null mutants lacking H3v, H4v, and base J, here I show that H4v is a major sign for transcription termination at TTSs. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J absence, accompanied by accumulation of sub-G1 population. Thus, I propose that the coordinated actions of H3v, H4v, and J provide compensatory mechanisms for each other in chromatin organization, transcription, replication, and cell-cycle progression.

scholarly journals Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei

2014 ◽  
Vol 42 (15) ◽  
pp. 9717-9729 ◽  
Author(s):  
David Reynolds ◽  
Laura Cliffe ◽  
Konrad U. Förstner ◽  
Chung-Chau Hon ◽  
T. Nicolai Siegel ◽  
...  
Keyword(s):  
Cell Death ◽  
Trypanosoma Brucei ◽  
Leishmania Major ◽  
Nuclear Dna ◽  
Epigenetic Modification ◽  
Transcription Termination ◽  
Gene Clusters ◽  
Regulation Of Transcription ◽  
Genome Wide ◽  
Rnap Ii

Abstract Base J, β-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase (RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.

scholarly journals CGGBP1-dependent CTCF-binding sites restrict ectopic transcription from SV40 early promoter

2020 ◽  
Author(s):  
Divyesh Patel ◽  
Manthan Patel ◽  
Subhamoy Datta ◽  
Umashankar Singh
Keyword(s):  
Binding Sites ◽  
Transcription Termination ◽  
Regulatory Protein ◽  
Ctcf Binding ◽  
Sv40 Promoter ◽  
Regulator Protein ◽  
Chromatin Regulator ◽  
Specific Manner ◽  
Direction Opposite

ABSTRACTBinding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones which show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription termination sequence required for the upstream transcription seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

scholarly journals Chromatin-Associated Protein Complexes Link DNA Base J and Transcription Termination in Leishmania

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Bryan C. Jensen ◽  
Isabelle Q. Phan ◽  
Jacquelyn R. McDonald ◽  
Aakash Sur ◽  
Mark A. Gillespie ◽  
...  
Keyword(s):  
Drug Therapy ◽  
Protein Complexes ◽  
Transcription Termination ◽  
Gene Clusters ◽  
Human Diseases ◽  
Effective Vaccine ◽  
Current Drug ◽  
Dna Base

Leishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids.

scholarly journals The Role of Cytoplasmic mRNA Cap-Binding Protein Complexes in Trypanosoma brucei and Other Trypanosomatids

Pathogens ◽  
2017 ◽  
Vol 6 (4) ◽  
pp. 55 ◽  
Author(s):  
Eden Freire ◽  
Nancy Sturm ◽  
David Campbell ◽  
Osvaldo de Melo Neto
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complexes ◽  
Binding Protein

scholarly journals Elucidating the Role of C/D snoRNA in rRNA Processing and Modification in Trypanosoma brucei

2007 ◽  
Vol 7 (1) ◽  
pp. 86-101 ◽  
Author(s):  
Sarit Barth ◽  
Boaz Shalem ◽  
Avraham Hury ◽  
Itai Dov Tkacz ◽  
Xue-hai Liang ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Gene Clusters ◽  
Snorna Gene ◽  
Small Nucleolar Rnas ◽  
Rrna Processing ◽  
Temperature Changes ◽  
Core Proteins ◽  
Processing Defects ◽  
Nucleolar Rnas

ABSTRACT Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2′-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2′-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.

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