scholarly journals Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei

2021 ◽  
Author(s):  
Tania Bishola Tshitenge ◽  
Lena Reichert ◽  
Bin Liu ◽  
Christine Clayton
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Untranslated Region ◽  
Rna Binding ◽  
Binding Protein ◽  
Developmental Regulation ◽  
Rna Binding Protein ◽  
Bloodstream Form ◽  
Tsetse Flies ◽  
Specific Gene

The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. In trypanosomes, gene expression regulation depends heavily on post-transcriptional mechanisms. Both the RNA-binding protein RBP10 and glycosomal phosphoglycerate kinase PGKC are expressed only in mammalian-infective forms. RBP10 targets procyclic-specific mRNAs for destruction, while PGKC is required for bloodstream-form glycolysis. Developmental regulation of both is essential: expression of either RBP10 or PGKC in procyclic forms inhibits their proliferation. We show that the 3′-untranslated region of the RBP10 mRNA is extraordinarily long - 7.3kb - and were able to identify six different sequences, scattered across the untranslated region, which can independently cause bloodstream-form-specific expression. The 3′-untranslated region of the PGKC mRNA, although much shorter, still contains two different regions, of 125 and 153nt, that independently gave developmental regulation. No short consensus sequences were identified that were enriched either within these regulatory regions, or when compared with other mRNAs with similar regulation, suggesting that more than one regulatory RNA-binding protein is important for repression of mRNAs in procyclic forms. We also identified regions, including an AT repeat, that increased expression in bloodstream forms, or suppressed it in both forms. Trypanosome mRNAs that encode RNA-binding proteins often have extremely extended 3′-untranslated regions. We suggest that one function of this might be to act as a fail-safe mechanism to ensure correct regulation even if mRNA processing or expression of trans regulators is defective.

scholarly journals A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

2020 ◽  
Vol 295 (42) ◽  
pp. 14291-14304
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine Clayton ◽  
Esteban Erben
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Critical Role ◽  
Rna Binding Protein ◽  
Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

The RNA-binding protein DRBD18 regulates processing and export of the mRNA encoding Trypanosoma brucei RNA-binding protein 10

2021 ◽  
Author(s):  
Tania Bishola Tshitenge ◽  
Bin Liu ◽  
Christine Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Gene Expression Regulation ◽  
Binding Protein ◽  
Mrna Export ◽  
Rna Binding Protein ◽  
Tsetse Flies ◽  
Nuclear Pore ◽  
Regulatory Sequences ◽  
Mrna Isoforms

The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. Trypanosome protein coding genes are arranged in polycistronic transcription units, so gene expression regulation depends heavily on post-transcriptional mechanisms. The essential RNA-binding protein RBP10 is expressed only in mammalian-infective forms, where it targets procyclic-specific mRNAs for destruction. We show that developmental regulation of RBP10 expression is mediated by the exceptionally long 7.3 Kb 3'-UTR of its mRNA. Different regulatory sequences that can independently enhance mRNA stability and translation in bloodstream forms, or destabilize and repress translation in procyclic forms, are scattered throughout the 3'-UTR. The RNA-binding protein DRBD18 is implicated in the export of a subset of mRNAs from the nucleus in procyclic forms. We confirmed that in bloodstream forms, DRBD18 copurifies the outer ring of the nuclear pore, mRNA export proteins and exon junction complex proteins. Loss of DRBD18 in bloodstream forms caused accumulation of several shortened RBP10 mRNA isoforms, with loss of longer species, but RNAi targeting the essential export factor MEX67 did not cause such changes, demonstrating specificity. Long RBP10 mRNAs accumulated in the nucleus, while shorter ones reached the cytoplasm. We suggest that DRBD18 binds to processing signals in the RBP10 3'-UTR, simultaneously preventing their use and recruiting mRNA export factors. DRBD18 depletion caused truncation of the 3'-UTRs of more than 100 other mRNAs, suggesting that it has an important role in regulating use of alternative processing sites.

scholarly journals Trypanosoma brucei RRM1 Is a Nuclear RNA-Binding Protein and Modulator of Chromatin Structure

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Arunasalam Naguleswaran ◽  
Kapila Gunasekera ◽  
Bernd Schimanski ◽  
Manfred Heller ◽  
Andrew Hemphill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Heat Shock ◽  
Trypanosoma Brucei ◽  
Chromatin Structure ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Content Type ◽  
Nuclear Rna

ABSTRACT TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

scholarly journals The RNA-binding protein RBP10 controls a regulatory cascade that defines bloodstream-form trypanosome identity

2016 ◽  
Author(s):  
Elisha Mugo ◽  
Christine Clayton
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Surface Protein ◽  
Bloodstream Form ◽  
Tsetse Flies ◽  
Regulatory Cascade ◽  
Gene Expression Control

AbstractGene expression control in the pathogen Trypanosoma brucei relies almost exclusively on post-transcriptional mechanisms, so RNA binding proteins must assume the burden that is usually borne by transcription factors. T. brucei multiply in the blood of mammals as bloodstream forms, and in the midgut of Tsetse flies as procyclic forms. We show here that a single RNA-binding protein, RBP10, defines the bloodstream-form trypanosome differentiation state. Depletion of RBP10 from bloodstream-form trypanosomes gives cells that can grow only as procyclic forms; conversely, expression of RBP10 in procyclic forms converts them to bloodstream forms. RBP10 binds to procyclic-specific mRNAs containing an UAUUUUUU motif, targeting them for translation repression and destruction. Products of RBP10 target mRNAs include not only the major procyclic surface protein and enzymes of energy metabolism, but also protein kinases and stage-specific RNA-binding proteins: consequently, alterations in RBP10 trigger a regulatory cascade.

scholarly journals Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

2021 ◽  
Vol 17 (6) ◽  
pp. e1009696
Author(s):  
Carla Gilabert Carbajo ◽  
Lucy J. Cornell ◽  
Youssef Madbouly ◽  
Zhihao Lai ◽  
Phillip A. Yates ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Feedback Mechanism ◽  
Bloodstream Form ◽  
Rna Recognition Motif ◽  
Intracellular Iron

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.

scholarly journals Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

2021 ◽  
Author(s):  
Carla Gilabert Carbajo ◽  
Lucy J Cornell ◽  
Youssef Madbouly ◽  
Zhihao Lai ◽  
Phillip A Yates ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Feedback Mechanism ◽  
Bloodstream Form ◽  
Rna Recognition Motif ◽  
Intracellular Iron

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.

scholarly journals The Interplay of RNA Binding Proteins, Oxidative Stress and Mitochondrial Dysfunction in ALS

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 552
Author(s):  
Jasmine Harley ◽  
Benjamin E. Clarke ◽  
Rickie Patani
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Mitochondrial Dysfunction ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Therapeutic Strategies ◽  
Lateral Sclerosis

RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.

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