scholarly journals Expression of normal levels of the Pumilio domain protein PUF3 is required for optimal growth of Trypanosoma brucei

2019 ◽  
Author(s):  
Kevin Kamanyi Marucha ◽  
Christine Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Ectopic Expression ◽  
Bloodstream Form ◽  
Fine Tuning ◽  
Growth Defect ◽  
Procyclic Form ◽  
Domain Protein

AbstractThe Trypanosoma brucei pumilio domain protein PUF3 is a cytosolic mRNA-binding protein that suppresses expression when tethered to a reporter mRNA. An induced reduction of PUF3 in bloodstream forms caused a slight growth defect and slightly delayed differentiation to the procyclic form, but the cells lost both defects upon prolonged cultivation. Both PUF3 genes could also be deleted in bloodstream-form and procyclic-form trypanosomes, suggesting that in vitro, at least, these life-cycle stages do not require PUF3. Procyclic forms without PUF3 grew somewhat slower than wild-type, but were able to transform to bloodstream forms after induced expression of the bloodstream-form RNA-binding protein RBP10. In contrast, ectopic expression of C-terminally tagged PUF3 in procyclic forms impaired viability. There was little evidence for specific binding of PUF3 to bloodstream-form mRNAs and RNAi had no significant effect on the transcriptome. Moreover, mass spectrometry revealed no PUF3 binding partners that might explain its suppressive activity. Since PUF3 is conserved in all Kinetoplastids, we suggest that it might be required within the invertebrate host, or perhaps implicated in fine-tuning gene expression.

Roles of the Pumilio domain protein PUF3 in Trypanosoma brucei growth and differentiation

Parasitology ◽  
2020 ◽  
Vol 147 (11) ◽  
pp. 1171-1183
Author(s):  
K. Kamanyi Marucha ◽  
C. Clayton
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Ectopic Expression ◽  
Fine Tuning ◽  
Growth Defect ◽  
Binding Studies ◽  
Procyclic Form ◽  
Delayed Differentiation ◽  
Domain Protein

AbstractTrypanosomes strongly rely on post-transcriptional mechanisms to control gene expression. Several Opisthokont Pumilio domain proteins are known to suppress expression when bound to mRNAs. The Trypanosoma brucei Pumilio domain protein PUF3 is a cytosolic mRNA-binding protein that suppresses expression when tethered to a reporter mRNA. RNA-binding studies showed that PUF3 preferentially binds to mRNAs with a classical Pumilio-domain recognition motif, UGUA[U/C]AUU. RNA-interference-mediated reduction of PUF3 in bloodstream forms caused a minor growth defect, but the transcriptome was not affected. Depletion of PUF3 also slightly delayed differentiation to the procyclic form. However, both PUF3 genes could be deleted in cultured bloodstream- and procyclic-form trypanosomes. Procyclic forms without PUF3 also grew somewhat slower than wild-type, but ectopic expression of C-terminally tagged PUF3 impaired their viability. PUF3 was not required for RBP10-induced differentiation of procyclic forms to bloodstream forms. Mass spectrometry revealed no PUF3 binding partners that might explain its suppressive activity. We conclude that PUF3 may have a role in fine-tuning gene expression. Since PUF3 is conserved in all Kinetoplastids, including those that do not infect vertebrates, we suggest that it might confer advantages within the invertebrate host.

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 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 TbUNC119 and Its Binding Protein Complex Are Essential for Propagation, Motility, and Morphogenesis of Trypanosoma brucei Procyclic Form Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (12) ◽  
pp. e15577 ◽  
Author(s):  
Shigeru Ohshima ◽  
Mitsuko Ohashi-Suzuki ◽  
Yutaka Miura ◽  
Yoshisada Yabu ◽  
Noriko Okada ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complex ◽  
Binding Protein ◽  
Procyclic Form

scholarly journals Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene

2007 ◽  
Vol 39 (4) ◽  
pp. 261-277 ◽  
Author(s):  
Pulak R Manna ◽  
Douglas M Stocco
Keyword(s):  
Transcriptional Repression ◽  
Protein Gene ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Ectopic Expression ◽  
Fine Tuning ◽  
Creb Binding Protein ◽  
Loss Of Function ◽  
Cis Element ◽  
Steroidogenic Acute Regulatory

AbstractTranscriptional regulation of the steroidogenic acute regulatory (StAR) protein gene by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP-response element (CRE; TGACGTCA) and is mediated by several sequence-specific transcription factors. We previously identified three CRE-like sites (within the −151/−1 bp cAMP-responsive region of the mouse StAR gene), of which the CRE2 site overlaps with an activator protein-1 (AP-1) motif (TGACTGA, designated as CRE2/AP-1) that can bind both CRE and AP-1 DNA-binding proteins. The present studies were aimed at exploring the functional crosstalk between CREB (CRE-binding protein) and cFos/cJun (AP-1 family members) on the CRE2/AP-1 element and its role in regulating transcription of the StAR gene. Using MA-10 mouse Leydig tumor cells, we demonstrate that the CRE and AP-1 families of proteins interact with the CRE2/AP-1 sequence. CREB, cFos, and cJun proteins were found to bind to the CRE2/AP-1 motif but not the CRE1 and CRE3 sites. Treatment with the cAMP analog (Bu)2cAMP augmented phosphorylation of CREB (Ser133), cFos (Thr325), and cJun (ser73). Chromatin immunoprecipitation studies revealed that the induction of CREB, cFos, and cJun by (Bu)2cAMP was correlated with protein–DNA interactions and recruitment of the coactivator CREB-binding protein (CBP) to the StAR promoter. EMSA studies employing CREB and cFos/cJun proteins demonstrated competition between these factors for binding to the CRE2/AP-1 motif. Transfection of cells containing the −151/−1 StAR reporter with CREB and cFos/cJun resulted in trans-repression of the StAR gene, an event tightly associated with CBP, demonstrating that both CREB and Fos/Jun compete with each other for binding with limited amounts of intracellular CBP. Overexpression of adenovirus E1A, which binds and inactivates CBP, markedly suppressed StAR gene expression. Ectopic expression of CBP eliminated the repression of the StAR gene by E1A and potentiated the activity of CREB and cFos/cJun on StAR promoter responsiveness. These findings identify molecular events involved in crosstalk between CREB and cFos/cJun, which confer both gain and loss of function on a single cis-element in fine-tuning of the regulatory events involved in transcription of the StAR gene.

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