Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

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.

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Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei

PLoS Pathogens ◽

10.1371/journal.ppat.1006560 ◽

2017 ◽

Vol 13 (8) ◽

pp. e1006560 ◽

Cited By ~ 34

Author(s):

Elisha Mugo ◽

Christine Clayton

Keyword(s):

Trypanosoma Brucei ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Bloodstream Form

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

10.1101/2021.11.29.470402 ◽

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.

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Faculty Opinions recommendation of Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717967962.793469395 ◽

2013 ◽

Author(s):

Gloria Rudenko

Keyword(s):

Trypanosoma Brucei ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Developmental Progression

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014346 ◽

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.

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Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Quarterly Reviews of Biophysics ◽

10.1017/s0033583515000207 ◽

2015 ◽

Vol 49 ◽

Cited By ~ 21

Author(s):

Marianna Teplova ◽

Thalia A. Farazi ◽

Thomas Tuschl ◽

Dinshaw J. Patel

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Hek293 Cells ◽

Structural Basis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Muscle Plasticity ◽

Rrm Domain

AbstractRNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

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KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei

RNA ◽

10.1261/rna.2244106 ◽

2006 ◽

Vol 12 (5) ◽

pp. 819-831 ◽

Cited By ~ 32

Author(s):

R. Salavati

Keyword(s):

Trypanosoma Brucei ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein

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The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression

Molecular Cell ◽

10.1016/j.molcel.2018.01.011 ◽

2018 ◽

Vol 69 (4) ◽

pp. 622-635.e6 ◽

Cited By ~ 28

Author(s):

Cindy Meyer ◽

Aitor Garzia ◽

Michael Mazzola ◽

Stefanie Gerstberger ◽

Henrik Molina ◽

...

Keyword(s):

Cell Cycle ◽

Stress Response ◽

Cell Cycle Progression ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Family ◽

Cycle Progression

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Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3494 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3494-3504 ◽

Cited By ~ 238

Author(s):

T D Levine ◽

F Gao ◽

P H King ◽

L G Andrews ◽

J D Keene

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Selection Procedure ◽

Vital Role ◽

Untranslated Regions ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Recognition Motifs

We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

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